Use Of Different Promoters Research Articles

Efficient lentiviral transduction method to gene modify cord blood CD8+ T cells for cancer therapy applications

Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. However, the limited numbers of autologous tumor-associated antigen (TAA)-specific T cells and the functional aberrancies, due to disease progression or treatment, remain factors that may significantly limit the success of the therapy. The use of allogeneic T cells, such as umbilical cord blood (CB) derived, overcomes these issues but requires gene modification to induce a robust and specific anti-tumor effect. CB T cells are readily available in CB banks and show low toxicity, high proliferation rates, and increased anti-leukemic effect upon transfer. However, the combination of anti-tumor gene modification and preservation of advantageous immunological traits of CB T cells represent major challenges for the harmonized production of T cell therapy products. In this manuscript, we optimized a protocol for expansion and lentiviral vector (LV) transduction of CB CD8+ T cells, achieving a transduction efficiency up to 83%. Timing of LV treatment, selection of culture media, and the use of different promoters were optimized in the transduction protocol. LentiBOOST was confirmed as a non-toxic transduction enhancer of CB CD8+ T cells, with minor effects on the proliferation capacity and cell viability of the T cells. Positively, the use of LentiBOOST does not affect the functionality of the cells, in the context of tumor cell recognition. Finally, CB CD8+ T cells were more amenable to LV transduction than peripheral blood (PB) CD8+ T cells and maintained a more naive phenotype. In conclusion, we show an efficient method to genetically modify CB CD8+ T cells using LV, which is especially useful for off-the-shelf adoptive cell therapy products for cancer treatment.

Modifications to the HIV-1 SAAVI MVA-C vaccine improve in vitro expression and in vivo immunogenicity

Two HIV-1 vaccines (SAAVI DNA-C2 and SAAVI MVA-C) were previously developed in South Africa and tested in preclinical studies and Phase 1 clinical trials. Here we report on improvements made to the SAAVI MVA-C vaccine design which include: the use of different promoters for both the Gag and Env genes, replacement of the native Gag gene with an in silico designed subtype C mosaic Gag antigen which forms virus-like particles and the modification of Env by sequence changes to improve stability and transport to the cell surface. A head-to-head comparison of the newly conceived MVAGD5 candidate vaccine with SAAVI MVA-C showed increased in vitro expression of both Env and Gag, and superior immunogenicity in rabbits. MVAGD5 induced high levels of binding antibodies to Env and Tier 1A and 1B neutralizing antibodies, neither of which were induced by SAAVI MVA-C.

Promoter and Terminator Optimization for DNA Methylation Targeting in Arabidopsis.

DNA methylation is an important epigenetic mark involved in gene regulation and silencing of transposable elements. The presence or absence of DNA methylation at specific sites can influence nearby gene expression and cause phenotypic changes that remain stable over generations. Recently, development of new technologies has enabled the targeted addition or removal of DNA methylation at specific sites of the genome. Of these new technologies, the targeting of the catalytic domain of Nicotiana tabacum DOMAINS REARRANGED METHYLTRANSFERASE 2 (ntDRM2cd) offers a promising tool for the addition of DNA methylation as it can directly methylate DNA. However, the methylation targeting efficiency of constructs using ntDRM2cd thus far has been relatively low. Previous studies have shown that the use of different promoters or terminators can greatly improve genome-editing efficiencies. In this study, we systematically survey a variety of promoter and terminator combinations to identify optimal combinations to use when targeting the addition of DNA methylation in Arabidopsis thaliana.

Multifaceted Role of PRDM Proteins in Human Cancer.

The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [Su(var)3-9, enhancer-of-zeste and trithorax] domain of histone methyltransferases (HMTs). These genes are involved in epigenetic regulation of gene expression through their intrinsic HMTase activity or via interactions with other chromatin modifying enzymes. In this way they control a broad spectrum of biological processes, including proliferation and differentiation control, cell cycle progression, and maintenance of immune cell homeostasis. In cancer, tumor-specific dysfunctions of PRDM genes alter their expression by genetic and/or epigenetic modifications. A common characteristic of most PRDM genes is to encode for two main molecular variants with or without the PR domain. They are generated by either alternative splicing or alternative use of different promoters and play opposite roles, particularly in cancer where their imbalance can be often observed. In this scenario, PRDM proteins are involved in cancer onset, invasion, and metastasis and their altered expression is related to poor prognosis and clinical outcome. These functions strongly suggest their potential use in cancer management as diagnostic or prognostic tools and as new targets of therapeutic intervention.

Heterologous expression of the ThIPK2 gene enhances drought resistance of common wheat

ThIPK2 is an inositol polyphosphate kinase gene cloned from Thellungiella halophila that participates in diverse cellular processes. Drought is a major limiting factor in wheat (Triticum aestivum L.) production. The present study investigated whether the application of the ThIPK2 gene could increase the drought resistance of transgenic wheat. The codon-optimized ThIPK2 gene was transferred into common wheat through Agrobacterium-mediated transformation driven by either a constitutive maize ubiquitin promoter or a stress-inducible rd29A promoter from Arabidopsis. Molecular characterization confirmed the presence of the foreign gene in the transformed plants. The transgenic expression of ThIPK2 in wheat led to significantly improve drought tolerance compared to that observed in control plants. Compared to the wild type (WT) plants, the transgenic plants showed higher seed germination rates, better developed root systems, a higher relative water content (RWC) and total soluble sugar content, and less cell membrane damage under drought stress conditions. The expression profiles showed different expression patterns with the use of different promoters. The codon-optimized ThIPK2 gene is a candidate gene to enhance wheat drought stress tolerance by genetic engineering.

Sumoylation of a small isoform of NFATc1 is promoted by PIAS proteins and inhibits transactivation activity

The NFAT family of transcription factors plays an important role in immune system development and function. NFATc1 and NFATc2 are highly expressed in peripheral T cells, and several isoforms are produced via the use of different promoters and polyadenylation sites. The specific isoforms with relatively long C-termini, NFATc1/C and NFATc2/A, have been shown to be modified by SUMO within their specific C-terminal regions, which regulates NFAT protein localization and transactivation activity. Here, we demonstrate that an isoform NFATc1/A, which has a short C-terminus and does not contain the sumoylation sites found in the long isoforms, is also modified by SUMO. NFATc1/A sumoylation increased with low level expression of SUMO E3 ligases, specifically PIAS1, PIAS3, and PIASy, in co-transfected cells. PIAS3 interacted with NFATc1/A and an active site mutant failed to promote NFATc1/A sumoylation, indicating a role for PIAS3 as a SUMO E3 ligase. A lysine residue at 351 within the central regulatory domain was identified as the major SUMO attachment site in both co-transfection and in vitro assays. Sumoylation of NFATc1/A did not affect nuclear translocation upon ionomycin and phorbol 12-myristate 13-acetate treatment. However, although sumoylation of NFATc1/A slightly increased protein stability, it inhibited transactivation activity for reporter genes driven by promoters containing NFAT sites. Our results indicate that the transactivation activity of NFATc1/A is negatively regulated by PIAS protein-mediated sumoylation, and that SUMO is a general regulator of NFAT family members with either long or short C-termini.

M39 - THE ASSOCIATION OF METHYLATION OF THE PROMOTER AND ANTIDEPRESSANTS IN TRANSCRIPTIONAL REGULATION OF THE BRAIN-DERIVED NEUROTROPHIC FACTOR EXON IV

Background Brain-Derived Neurotrophic Factor (BDNF) is important for the survival of existing neurons and promotion of the growth and differentiation of new neurons and synapses. BDNF protein expression has been associated with various psychiatric disorders including major depressive disorders. The gene contains eight exons with their individual promoters that initiate transcription of multiple mRNA transcripts, each of which contains an alternative 5′ exon spliced to a common 3′ coding exon IX. The use of different promoters allows a diverse, temporal and spatial regulation of the gene. We were particularly interested in exon IV as it has been shown to play an important role in response to monoaminergic anti-depressant drugs which mainly exert their effects via downstream signaling leading to phosphorylation of CREB (calcium/cAMP response element binding protein) and possibly a transcriptional repressor, MeCP2. The pCREB binds to the response element in the promoter region and enhances exon IV transcription. Our aim is to investigate the interaction between antidepressant-mediated transcriptional regulation and BNDF exon IV methylation. Methods We treated the SH-SY5Y neuroblastoma cell line with Venlafaxine, Fluoxetine, and Mirtazapine and standardized the concentrations and time points at which these drugs increase the phosphorylation of CREB as well as MeCP2 using western blot analysis. We cloned BDNF exon IV promoter region upstream of the luciferase gene in a CpG free reporter vector and reporter assays were performed in the presence of Venlafaxine, Fluoxetine, and Mirtazapine at desired concentrations. Immunofluorescence was used to study the drug-mediated changes in localization of MeCP2 and CREB (phosphorylated and unphosphorylated). We used site-directed mutagenesis to replace the CpGs of interest in the reporter construct by methylated CpGs. Results An increased phosphorylation of both CREB and MeCP2 as early as 0, 5 h and up to 2hrs treatment of SHSY-5Y cells by Venlafaxine (10 μM /50 μM), Fluoxetine (5 μM/10 μM) and Mirtazapine (10 μM /50 μM) was observed. We did not see any increase in the exonIV transcriptional activity under these conditions in the unmethylated plasmid whereas methylated plasmid showed a statistically significant upregulation with all the three drugs. Immunofluorescence data shows that Fluoxetine changes the localization of only unphosphorylated MeCP2 to nucleo-cytoplasmic from nuclear. Venlafaxine and Mirtazapine showed no change in localization of MeCP2. Discussion A previous study from our group has shown that methylation of the CpG residues present on exon IV promoter complements the increase in serum BDNF levels by Venlafaxine and Fluoxetine, at physiological doses. The response to the drugs can be predicted by the methylation of the CpGs present in the proximal promoter, -35 bp to -147 bp. We hypothesize, kinases that get activated downstream of the 5-HT receptor, phosphorylate methyl binding protein (MeCP2) and thus abrogate its binding to the methylated CPGs. Contrary to MeCP2, phosphorylated MeCP2 can bind to unmethylated CpGs. As MeCP2 is a transcriptional repressor, it counteracts the upregulation of BDNF exon IV transcript by pCREB binding and thereby determines the efficacy of the drugs. This work commences a profound understanding of the epigenetic regulation of exon IV by antidepressants. Furthermore, it emerges to be in support with previous clinical findings regarding the use of CPG methylation as a predictive marker for antidepressant therapy.

Translational control of tropomyosin expression in vertebrate hearts.

The tropomyosin (TM) gene family produces a set of related TM proteins with important functions in striated and smooth muscle, and nonmuscle cells. In vertebrate striated muscle, the thin filament consists largely of actin, TM, the troponin (Tn) complex (Tn-I, Tn-C and Tn-T), and tropomodulin (Tmod) and is responsible for mediating Ca(2+) control of muscle contraction and relaxation. There are four known genes (designated as TPM1, TPM2, TPM3, and TPM4) for TM in vertebrates. The four TM genes generate a multitude of tissue- and developmental-specific isoforms through the use of different promoters, alternative mRNA splicing, different 3'-end mRNA processing and tissue-specific translational control. In this review, we have focused mainly on the regulation of TM expression in striated muscles, primarily in vertebrate hearts with special emphasis on translational control using mouse and Mexican axolotl animal models.

Comparative breast cancer research, lessons from companion animals.

Comparative breast cancer research, lessons from companion animals.

TAp73β-mediated suppression of cell migration requires p57Kip2 control of actin cytoskeleton dynamics

The TP73 gene, a member of the p53 family, due to the use of different promoters and alternative splicing, is transcribed into different isoforms with contrasting attributes and which contribute to its functional diversity. Considerable efforts are made to identify the functional diversity of the p73 splicing variants during tumorigenesis. TAp73α and TAp73β isoforms have been shown to differentially regulate cell cycle progression, differentiation and apoptosis. Interestingly, a particular increase in expression of the TAp73 isoform, in favor of the α splicing variant, has been reported in multiple tumour types. Here, we report a distinctive role for TAp73β isoform in the control of cell migration and invasion. In fact, TAp73β- dependent induction of p57(Kip2) expression accounted for inhibitory effects on the actin cytoskeleton dynamics and thereby cancer cell motility. In contrast, TAp73α is not able to induce p57(Kip2) expression, and exhibits a positive effect on actin cytoskeleton dynamics as well as cell migration and invasion. In conclusion, the inhibitory effect on cell migration and invasion of TAp73β would qualify this distinct p73 isoform as tumor suppressor gene. In contrast, the promoting effect of TAp73α on cell motility and invasion strengthens the potential oncogenic activities of this p73 isoform.

Progesterone Receptor Isoforms in the Mammary Gland of Cats and Dogs

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

Phosphodiesterase 4 regulates the migration of B16-F10 melanoma cells

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. Eleven PDE gene families (PDE1-11) have been identified and several PDE isoforms are selectively expressed in various cell types. PDE4 family members specifically hydrolyze cyclic AMP (cAMP). Four genes (PDE4A-D) are known to encode PDE4 enzymes, with additional diversity generated by the use of alternative mRNA splicing and the use of different promoters. While PDE4 selective inhibitors show therapeutic potential for treating major diseases such as asthma and chronic obstructive pulmonary disease, little is known concerning the role of PDE4 in malignant melanoma. In this study, we examined the role of PDE4 in mouse B16-F10 melanoma cells. In these cells, PDE4 activity was found to be ∼60% of total PDE activity. RT-PCR detected only PDE4B and PDE4D mRNA. Cell growth was inhibited by the cAMP analog, 8-bromo-cAMP, but not by the specific PDE4 inhibitors, rolipram and denbufylline, which increased intracellular cAMP concentrations. Finally, migration of the B16-F10 cells was inhibited by the PDE4 inhibitors and 8-bromo-cAMP, while migration was increased by a protein kinase A (PKA) inhibitor, PKI(14-22), and was not affected by 8-pCPT-2'-O-Me-cAMP, which is an analog of exchange protein activated by cAMP (Epac). The inhibitory effect of rolipram on migration was reversed by PKI(14-22). Based on these results, PDE4 appears to play an important role in the migration of B16-F10 cells, and therefore may be a novel target for the treatment of malignant melanoma.

Involvement of transcription factor GATA-4 in regulation of CYP19 gene during folliculogenesis and luteinization in buffalo ovary

CYP19 gene encode aromatase, the key enzyme of estrogen biosynthesis, is regulated in species- and tissue-specific manner by alternate use of different promoters. Previously, we have reported the cloning and characterization of tissue-specific promoter and transcripts in buffalo ovary and placenta. In human and rat ovary, FSH induces the phosphorylation of transcription factor CREB (cAMP response element binding protein) through PKA pathway which binds to cAMP response element like sequence (CLS) in CYP19 gene ovarian promoter. However, in buffalo as well as in bovine, in silico analysis of ovary specific promoter sequence identified a single base pair deletion in CLS site and is designated as CLS-like sequence. To understand if CLS with a point mutation is still a functional cis-element and is involved in FSH stimulated transactivation of CYP19 gene in buffalo ovary, the present study was thus aimed to functionally characterize the role of buffalo CLS in CYP19 gene transactivation. We also studied the involvement of GATA-4, having consensus binding sites in CYP19 gene ovarian promoter in the vicinity of CLS during different stages of the buffalo estrus cycle. Reporter construct analyses and EMSA results showed that CLS is playing no significant role in CYP19 gene regulation in buffalo ovary. Real time absolute quantification of GATA-4 showed the differential expression of GATA-4 mRNA during folliculogenesis and luteinization with significantly higher transcript abundance in large follicle in comparison to other tissues. Western blot analysis of granulosa cells nuclear protein isolated from different stage of follicular development (small and large follicles) and differentiation (corpus luteum) showed that abundance of phosphorylated GATA-4 (Ser261) was significantly higher in granulosa cell nuclear protein of large follicles as compared to small follicles and corpora lutea. Interestingly, binding studies using ChIP showed significantly enhanced binding to the CYP19 gene promoter in large follicle which was seen to be declined in the luteal tissue. Similar results were obtained in the in vitro experiments as well. Finally, RNAi experiments were performed to validate the involvement of GATA-4 in CYP19 gene regulation. Results of RNAi showed that knockdown of GATA-4 mRNA significantly declined CYP19 gene mRNA as well as 17β-estradiol contents. In conclusion, result of the present study indicated that that in the absence of consensus CRE (cAMP response element); GATA-4 could be a downstream effector of cAMP/PKA pathway in regulation of CYP19 gene during folliculogenesis and luteinization.

Do We Know the Complete Story of TPM1κ Expression in Vertebrate Hearts?

Copyright: © 2012 Siddiqui MT. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Tropomyosin (TM) is a component of myofibrils, the contractile apparati of striated muscle cells. The assembly of a myofibril involves the precise ordering of several proteins into a linear array of sarcomeres. The process is complicated further because most of these proteins have multiple isoforms. Recent demonstration of the association of TM mutations with Hypertrophic Cardiomyopathy (HCM)/Familial Hypertrophic Cardiomyopathy (FHC) [1-3] or other cardiomyopathies such as Dilated Cardiomyopathy (DCM) [4,5] and nemalin skeletal myopathy [6] in humans have spurred renewed interest in the structural/functional relationships of TM. In vertebrate striated muscle, the thin filament consists largely of actin, TM, the Troponin (Tn) complex (Tn-I, Tn-C and Tn-T), Tropomodulin (Tmod), and Leiomodin (Lmod). It is responsible for mediating Ca+2 control of contraction and relaxation. With the influx of Ca+2 in muscle cells, troponin-C binds with Ca+2 and undergoes a rapid conformational change, which causes additional conformation changes in the TM-Tn complex exposing the myosin associated ATPase activity. As a result, the free myosin associated ATPase activity hydrolyzes ATP with the release of energy that helps muscle to contract. There are four genes (designated as TPM1, TPM2, TPM3, and TPM4) for TM in vertebrates [7,8] except for zebrafish where there are six TM genes [9], which generate a multitude of tissue and developmental specific isoforms through the use of different promoters, alternative mRNA splicing, and tissue specific translational control [10]. The functional significance of this diversity and how it may affect interactions with other sarcomeric proteins is not fully understood. Sarcomeric isoforms for both TPM1 and TPM2 are expressed in large mammals, including humans [11]. The protein of the sarcomeric isoform of TPM3 is not expressed in mammalian hearts. In mammals TPM4 is truncated relative to the gene found in avian [12] and amphibian species [13].

Methylseleninic acid is a novel suppressor of aromatase expression

Elevated circulating estrogen levels, as a result of increased peripheral aromatization of androgens by aromatase, have been indicated to underlie the association between obesity and a higher risk of breast cancer in postmenopausal women. Although aromatase inhibitors have been used as a first-line therapy for estrogen receptor-positive breast cancer in postmenopausal women, their potential as breast cancer chemopreventive agents has been limited due to toxicities and high costs. It is therefore imperative to develop new aromatase-inhibiting/suppressing agents with lower toxicities and lower costs for breast cancer chemoprevention, especially in obese postmenopausal women. The expression of the aromatase gene, CYP19, is controlled in a tissue-specific manner by the alternate use of different promoters. In obese postmenopausal women, increased peripheral aromatase is primarily attributed to the activity of the glucocorticoid-stimulated promoter, PI.4, and the cAMP-stimulated promoter, PII. In the present study, we show that methylseleninic acid (MSA), a second-generation selenium compound, can effectively suppress aromatase activation by dexamethasone, a synthetic glucocorticoid, and forskolin, a specific activator of adenylate cyclase. Unlike the action of aromatase inhibitors, MSA suppression of aromatase activation is not mediated via direct inhibition of aromatase enzymatic activity. Rather, it is attributable to a marked downregulation of promoters PI.4- and PII-specific aromatase mRNA expression, and thereby a reduction of aromatase protein. Considering the low-cost and low-toxicity nature of MSA, our findings provide a strong rationale for the further development of MSA as a breast cancer chemopreventive agent for obese postmenopausal women.

Tissue-specific promoter methylation and histone modification regulate CYP19 gene expression during folliculogenesis and luteinization in buffalo ovary

Aromatase, the key enzyme of estrogen biosynthesis, is encoded by the CYP19 gene. The expression of CYP19 gene is regulated in species- and tissue-specific manner by alternate use of different promoters. We have previously, cloned and characterized the tissue-specific promoter and tissue-specific transcripts in preovulatory (granulosa cells) and postovulatory (corpus luteum) structure of buffalo ovary. The present study was aimed to understand if epigenetic gene regulation through DNA methylation and histone modifications is involved in tissue-specific CYP19 gene regulation during folliculogenesis and luteinization in buffalo ovary. Methylation analysis of five CpG dinucleotides of ovary specific proximal promoter II showed hypo-methylation in large follicle while hyper-methylation in corpus luteum. However, PI.1, the exclusive promoter responsible for residual CYP19 gene expression in corpus luteum, was found to be hypermethylated. Analysis of histone modifications using ChIP assay revealed that the distal promoter (PI.1) of CYP19 gene is ∼40-fold more enriched with acetylated Histone H3 in corpus luteum than in the large follicle. This indicates that PI.1 chromatin was more accessible for transcription in corpus luteum as compared to large follicles. The chromatin accessibility for the proximal promoter (PII) in the preovulatory stage tends to be higher than the luteal tissue. However, the difference was not found to be significant. In vitro experiments showed the similar results. In conclusion, results of the present study suggests that tissue-specific methylation status of PII and chromatin remodeling through histone modifications of PI.1, coincides with the changes in expression of CYP19 gene and thus are the regulatory mechanism controlling its tissue-specific expression and promoter activity during folliculogenesis and luteinization.

Progress of tissue culture and genetic transformation research in pigeon pea [Cajanus cajan (L.) Millsp.

Pigeon pea [Cajanus cajan (L.) Millsp.] (Family: Fabaceae) is an important legume crop cultivated across 50 countries in Asia, Africa, and the Americas; and ranks fifth in area among pulses after soybean, common bean, peanut, and chickpea. It is consumed as a major source of protein (21%) to the human population in many developing countries. In India, it is the second important food legume contributing to 80% of the global production. Several biotic and abiotic stresses are posing a big threat to its production and productivity. Attempts to address these problems through conventional breeding methods have met with partial success. This paper reviews the chronological progress made in tissue culture through organogenesis and somatic embryogenesis, including the influence of factors such as genotypes, explant sources, and culture media including the supplementation of plant growth regulators. Comprehensive lists of morphogenetic pathways involved in in vitro regeneration through organogenesis and somatic embryogenesis using different explant tissues of diverse pigeon pea genotypes are presented. Similarly, the establishment of protocols for the production of transgenics via particle bombardment and Agrobacterium-mediated transformation using different explant tissues, Agrobacterium strains, Ti plasmids, and plant selectable markers, as well as their interactions on transformation efficiency have been discussed. Future research thrusts on the use of different promoters and stacking of genes for various biotic and abiotic stresses in pigeon pea are suggested.

Conquering the complex world of human septins: implications for health and disease

Septins are highly conserved filamentous proteins first characterized in budding yeast and subsequently identified in must eukaryotes. Septins can bind and hydrolyze GTP, which is intrinsically related to their formation of septin hexamers and functional protein interactions. The human septin family is composed of 14 loci, SEPT1-SEPT14, which encode dozens of different septin proteins. Their central GTPase and polybasic domain regions are highly conserved but they diverge in their N-terminus and/or C-terminus. The mechanism by which the different isoforms are generated is not yet well understood, but one can hypothesize that the use of different promoters and/or alternative splicing could give rise to these variants. Septins perform diverse cellular functions according to tissue expression and their interacting partners. Functions identified to date include cell division, chromosome segregation, protein scaffolding, cellular polarity, motility, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, and DNA damage response. Their expression is tightly regulated to maintain proper filament assembly and normal cellular functions. Alterations of these proteins, by mutation or expression changes, have been associated with a variety of cancers and neurological diseases. The association of septins with cancer results from alterations of expression in solid tumors or translocations in leukemias [mixed lineage leukemia (MLL)]. Expression changes in septins have also been associated with neurological conditions such as Alzheimer's and Parkinson's disease, as well as retinopathies, hepatitis C, spermatogenesis and Listeria infection. Pathogenic mutations of SEPT9 were identified in the autosomal dominant neurological disorder hereditary neuralgic amyotrophy (HNA). Human septin research over the past decade has established their importance in cell biology and human disease. Further functional characterization of septins is crucial to our understanding of their possible diagnostic, prognostic, and therapeutic applications.

Characterization of the Human Insulin-induced Gene 2 (INSIG2) Promoter: THE ROLE OF Ets-BINDING MOTIFS

Insulin-induced gene 2 (INSIG2) and its homolog INSIG1 encode closely related endoplasmic reticulum proteins that regulate the proteolytic activation of sterol regulatory element-binding proteins, transcription factors that activate the synthesis of cholesterol and fatty acids in animal cells. Several studies have been carried out to identify INSIG2 genetic variants associated with metabolic diseases. However, few data have been published regarding the regulation of INSIG2 gene expression. Two Insig2 transcripts have been described in rodents through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Herein we report the cloning and characterization of the human INSIG2 promoter and the detection of an INSIG2-specific transcript homologous to the Insig2b mouse variant in human liver. Deletion analyses on 3 kb of 5'-flanking DNA of the human INSIG2 gene revealed the functional importance of a 350-bp region upstream of the transcription start site. Mutated analyses, chromatin immunoprecipitation assays, and RNA interference analyses unveiled the significance of an Ets-consensus motif in the proximal region and the interaction of the Ets family member SAP1a (serum response factor (SRF) accessory protein-1a) with this region of the human INSIG2 promoter. Moreover, our findings suggest that insulin activated the human INSIG2 promoter in a process mediated by phosphorylated SAP1a. Overall, these results map the functional elements in the human INSIG2 promoter sequence and suggest an unexpected regulation of INSIG2 gene expression in human liver.

Regulation of FGF1 Gene Promoter through Transcription Factor RFX1

Fibroblast growth factor 1 (FGF1) has been suggested to have an important role in cell growth, proliferation, and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven green fluorescence (F1BGFP) has been shown to monitor endogenous FGF1 expression. F1BGFP could also be used to isolate neural stem/progenitor cells from embryonic, neonatal, and adult mouse brains or to isolate glioblastoma stem cells (GBM-SCs) from human glioblastoma tissues. Here, we present evidence that transcription factor RFX1 could bind the 18-bp cis-elements (−484 to −467) of the F1B promoter, modulate F1BGFP expression and endogenous FGF1 expression, and further regulate the maintenance of GBM-SCs. These observations were substantiated by using yeast one-hybrid assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, gain- and loss-of-function assays, and neurosphere assays. Overexpression of RFX1 was shown to down-regulate FGF-1B mRNA expression and neurosphere formation in human glioblastoma cells, whereas RNA interference knockdown of RFX1 demonstrated the opposite effects. Our findings provide insight into FGF1 gene regulation and suggest that the roles of FGF1 and RFX1 in the maintenance of GBM-SCs. RFX1 may negatively regulate the self-renewal of GBM-SCs through modulating FGF-1B and FGF1 expression levels by binding the 18-bp cis-elements of the F1B promoter.