Decrease In Transcriptional Activity Research Articles

The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter.

Pathological expansion of a G4C2 repeat, located in the 5' regulatory region of C9orf72, is the most common genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). C9orf72 patients have highly variable onset ages suggesting the presence of modifying factors and/or anticipation. We studied 72 Belgian index patients with FTLD, FTLD–ALS or ALS and 61 relatives with a C9orf72 repeat expansion. We assessed the effect of G4C2 expansion size on onset age, the role of anticipation and the effect of repeat size on methylation and C9orf72 promoter activity. G4C2 expansion sizes varied in blood between 45 and over 2100 repeat units with short expansions (45–78 units) present in 5.6% of 72 index patients with an expansion. Short expansions co-segregated with disease in two families. The subject with a short expansion in blood but an indication of mosaicism in brain showed the same pathology as those with a long expansion. Further, we provided evidence for an association of G4C2 expansion size with onset age (P<0.05) most likely explained by an association of methylation state of the 5' flanking CpG island and expansion size in blood (P<0.0001) and brain (P<0.05). In several informative C9orf72 parent–child transmissions, we identified earlier onset ages, increasing expansion sizes and/or increasing methylation states (P=0.0034) of the 5' CpG island, reminiscent of disease anticipation. Also, intermediate repeats (7–24 units) showed a slightly higher methylation degree (P<0.0001) and a decrease of C9orf72 promoter activity (P<0.0001) compared with normal short repeats (2–6 units). Decrease of transcriptional activity was even more prominent in the presence of small deletions flanking G4C2 (P<0.0001). Here we showed that increased methylation of CpGs in the C9orf72 promoter may explain how an increasing G4C2 size lead to loss-of-function without excluding repeat length-dependent toxic gain-of-function. These data provide insights into disease mechanisms and have important implications for diagnostic counseling and potential therapeutic approaches.

Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

KAL1 is implicated in 5% of Kallmann syndrome cases, a disorder which genotypically overlaps with septo-optic dysplasia (SOD). To date, a reporter-based assay to assess the functional consequences of KAL1 mutations is lacking. We aimed to develop a luciferase assay for novel application to functional assessment of rare KAL1 mutations detected in a screen of 422 patients with SOD.Quantitative analysis was performed using L6-myoblasts stably expressing FGFR1, transfected with a luciferase-reporter vector containing elements of the FGF-responsive osteocalcin promoter.The two variants assayed [p.K185N, p.P291T], were detected in three females with SOD (presenting with optic nerve hypoplasia, midline and pituitary defects). Our novel assay revealed significant decreases in transcriptional activity [p.K185N: 21% (p < 0.01); p.P291T: 40% (p < 0.001)].Our luciferase-reporter assay, developed for assessment of KAL1 mutations, determined that two variants in females with hypopituitarism/SOD are loss-of-function; demonstrating that this assay is suitable for quantitative assessment of mutations in this gene.

Effect of packaging materials, environmental factors and rRNA transcriptional activity of surface microflora on red-smear cheese defect development

Two Swiss cheese varieties were investigated to elucidate how microbial growth and rRNA transcriptional activity leads to a defective cheese surface when red-smear cheese portions are wrapped in plastic films. Four alternative films with different water and gas permeabilities were used for packaging experiments. A defective smear was associated with anaerobic conditions, increased water activity and decreased pH over a period of 8 weeks at 8 °C. Decreases of log 0.7–1.6 total bacteria, log 0.6–1.2 Corynebacterium casei and log 0.6–1.6 Bacillus spp. 16S rRNA copy numbers measured by RT-qPCR were obtained for both varieties under packaging conditions, suggesting a decrease in transcriptional activity. One alternative film, porous to O2, CO2 and water vapour, created a less humid smear and higher pH during storage. Cheese smear development mainly depended upon the permeability of the packaging film, and was associated with a decrease in rRNA transcriptional activity of cheese smear microorganisms.

Increased Electrophoretic Mobility of Long-Type GATA-6 Transcription Factor upon Substitution of Its PEST Sequence

The transcriptional factor GATA-6 gene produces two translational isoforms from a single mRNA through ribosomal leaky scanning. L-type GATA-6 has an extension of 146 amino acid residues at its amino terminus. In the extension, there is a unique PEST sequence (Glu31-Cys46), which is composed of an amino terminal Pro-rich segment and a carboxyl terminal Ser-cluster. Substitution of either half of the PEST sequence with Ala residues by cassette mutagenesis reduced the apparent molecular size of L-type GATA-6 on SDS-polyacrylamide gel-electrophoresis. However, the effect of substitution of the Pro-rich segment was much more significant; the mobility increase of the Pro-rich segment on the gel was 13% while that of the Ser-cluster was 8%. Substitution of each amino acid residue demonstrated that the effect of Pro substitution is greater than that of the Ser and Thr residues. Such increased mobility of L-type GATA-6 in the presence of a detergent may apparently correlate with the decrease in transcription activity in vivo as determined by means of luciferase reporter gene assay. The activity of ΔAla (with Ala residues instead of the PEST sequence) was reduced to one fifth of that of ΔA (with the PEST sequence). These results suggest that the PEST sequence of L-type GATA-6 does not function as a constitutive protein degradation signal, but rather plays structural and functional roles in the activation of gene expression on the GATA responsive promoter.

Abstract 200: Cardiomyocyte-specific Deletion Of Rhoa Accelerates Heart Failure But Prevents Cardiac Fibrosis In Response To Pressure-overload

Heart failure is a progressive disease characterized by loss of cardiac contractility, interstitial fibrosis, and chamber remodeling. Though several signaling pathways have been identified, the precise mechanisms involved in regulating pathological cardiac responses remain unclear. The Ras-related small G protein RhoA is an identified mediator of in vivo pathological hypertrophy. However, it also promotes cell survival and is cardioprotective after ischemia/repurfusion injury. Therefore, to determine the molecular mechanisms that underlie these opposing roles for RhoA in the myocardium, we generated mice with cardiomyocyte-specific deletion of RhoA (RhoAfl/fl-αMHC-Cre). As compared to wildtype mice, hearts from RhoAfl/fl-αMHC-Cre mice showed similar functional, structural, and growth parameters, both at baseline and in response to acute transverse aortic constriction (TAC) (2wks), suggesting RhoA plays no major role in either homeostatic or acute stress-induced cardiac conditions. However, after exposure to chronic TAC for 8wks, hearts from RhoAfl/fl-αMHC-Cre mice developed an accelerated dilation, with significant loss of contractile function. Mechanistically, cardiomyocyte-specific loss of RhoA reduced ERK1/2 activation and perturbed agonist-evoked calcium cycling, through aberrant IP3 and DAG signaling, indicating that RhoA mediates its cardioprotective effects through regulation of these pathways. Despite this, and surprisingly, hearts from RhoAfl/fl-αMHC mice also showed significantly decreased TAC-induced cardiac fibrosis, with a demonstrated decrease in transcriptional activation of genes involved in the fibrotic response, including the serum response factor (SRF), as well as the secreted factor BMP7. Taken together, our data reveal an interesting, yet highly complex signaling and regulatory mechanism for RhoA, whereby RhoA signaling is both cardioprotective against development of heart failure in response to chronic stress, but also cardio-deleterious through its capacity to activate pro-fibrotic genes.

Mechanisms Mediating the Effects ofγ-Tocotrienol When Used in Combination with PPARγAgonists or Antagonists on MCF-7 and MDA-MB-231 Breast Cancer Cells

γ-Tocotrienol is a natural vitamin E that displays potent anticancer activity, and previous studies suggest that these effects involve alterations in PPARγ activity. Treatment with 0.5–6 μM γ-tocotrienol, 0.4–50 μM PPARγ agonists (rosiglitazone or troglitazone), or 0.4–25 μM PPARγ antagonists (GW9662 or T0070907) alone resulted in a dose-responsive inhibition of MCF-7 and MDA-MB-231 breast cancer proliferation. However, combined treatment of 1–4 μM γ-tocotrienol with PPARγ agonists reversed the growth inhibitory effects of γ-tocotrienol, whereas combined treatment of 1–4 μM γ-tocotrienol with PPARγ antagonists synergistically inhibited MCF-7 and MDA-MB-231 cell growth. Combined treatment of γ-tocotrienol and PPARγ agonists caused an increase in transcription activity of PPARγ along with increased expression of PPARγ and RXR, and decrease in PPARγ coactivators, CBP p/300, CBP C-20, and SRC-1, in both breast cancer cell lines. In contrast, combined treatment of γ-tocotrienol with PPARγ antagonists resulted in a decrease in transcription activity of PPARγ, along with decreased expression of PPARγ and RXR, increase in PPARγ coactivators, and corresponding decrease in PI3K/Akt mitogenic signaling in these cells. These findings suggest that elevations in PPARγ are correlated with increased breast cancer growth and survival, and treatment that decreases PPARγ expression may provide benefit in the treatment of breast cancer.

Transcriptional regulation of skeletal muscle GLUT4 by elevated long chain fatty acids

A high-fat diet plays a role in insulin resistance and suppresses the expression of the glucose transporter 4 (GLUT4) in skeletal muscle. The objective of this study was to determine the effect of the individual long chain fatty acids, saturated or unsaturated, cis- or trans-confirmation, on transcriptional activity of the human GLUT4 promoter. We used a full-length human GLUT4 promoter with a luciferase reporter transfected into rat skeletal muscle (L6) cells. Transfected myotubes were exposed to individual long chain fatty acids (16–20 carbon), both saturated and unsaturated (one and two points of unsaturation), and 18:1 cis- and trans-confirmation. All fatty acids tested either had no effect on transcriptional activity or significantly reduced transcriptional activity. We found a decrease in transcriptional activity with two of the long-chain saturated fatty acids. Neither the cis- and trans-confirmation fatty acid regulated the transcriptional activity of the GLUT4 promoter similarly nor did the monounsaturated and polyunstaturated fatty acid regulate the transcriptional activity of the GLUT4 promoter similarly. We conclude that long chain fatty acids differentially regulate the expression of GLUT4 in muscle and any alteration by these particular fatty acids in expression of GLUT4 would result in a deleterious effect on glucose transport. This research was supported by East Carolina University.

The Crucial Role of GATA-1 in CCR3 Gene Transcription: Modulated Balance by Multiple GATA Elements in the CCR3 Regulatory Region

GATA-1, a zinc finger-containing transcription factor, regulates not only the differentiation of eosinophils but also the expression of many eosinophil-specific genes. In the current study, we dissected CCR3 gene expression at the molecular level using several cell types that express varying levels of GATA-1 and CCR3. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1. A reporter plasmid assay showed that constructs harboring exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of endogenous GATA-1. Introduction of a dominant-negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1 bound to virtually all seven putative GATA elements present in exon 1-intron 1, the first GATA site in exon 1 exhibited the highest binding affinity for GATA-1 and was solely responsible for GATA-1-mediated transactivation. The fourth and fifth GATA sites in exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1-mediated transcription of eosinophil-specific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore, mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the CCR3 gene.

Idiopathic CD4 Lymphocytopenia Due to Loss of Heterozygosity of the Mutant CD2 Gene

AbstractAbstract 2774Idiopathic CD4 lymphocytopenia (ICL) is defined as persistent depression of CD4+ T cells with the absence of any known causes including HIV infection or drugs. Although sporadic case reports of ICL suggest heterogeneity of the disease, its mechanisms remain largely unknown. We experienced a case of ICL with decreased CD2 expression that was indicated to be due to loss of heterozygosity (LOH) of CD2 gene. A 40-year-old previously healthy man presented with dry cough and high fever. He was diagnosed as pneumonia which turned out to be Pneumocystis carinii infection by the PCR examination of bronchoalveolar lavage fluid. Ultrasonographic screening revealed multiple liver tumors, and fine needle aspiration biopsy of the liver led to the diagnosis of diffuse large B-cell lymphoma. He soon developed systemic lymphadenopathy and brain tumors, and histological examination of inguinal lymph node revealed Hodgkin lymphoma. In spite of the discrepant histological diagnoses of the liver and the inguinal lymph node, both lymphoma cells were shown to be positive for EBER-ISH. Flow cytometric analysis of his peripheral blood cells revealed a significant decrease in CD3+ T-cell counts (111 cells/μl) with 32 cells/μl CD4+ T cells, although he was negative for HIV infection. Of note, CD2 expression was severely diminished on both T and NK cells, and more than half of TCRαβ positive T cells were CD4/CD8 double negative. No cell clonality was detected by TCR gene rearrangement analysis, indicating that these T cells with abnormal phenotypes constitute a polyclonal population. Thus both pneumonia and EBV-associated lymphomas were considered to be opportunistic complications of T-cell immunodeficiency. He was given 2 courses of chemotherapy and whole brain irradiation, but these treatments resulted in poor response. He finally received allogeneic bone marrow transplantation from an HLA-matched unrelated donor, and his tumors gradually decreased with successful engraftment of donor cells with normal CD2 expression. CD2 is a glycoprotein that mediates adhesion of T cells to antigen presenting cells and provides a positive costimulatory signal for TCR-mediated activation of T cells. Decreased CD2 expression has previously been documented in 2 cases of ICL (J Clin Immunol 1994;14:359, Ann Allergy Asthma Immunol 2006;98:294), but its pathogenesis has not been clarified. To elucidate the immunomodulatory effect of CD2 deficiency, we performed a functional assay of the patient's native T cells by stimulating with PMA and ionomycin. They showed biased cytokine production with excessive Th2 and suppressed Th1 responses. We established an EBV-immortalized lymphoblastoid B-cell line (LCL) from a healthy volunteer and examined autologous T-cell activity against LCL with or without the presence of CD2-blocking antibody. Blocking of CD2 signaling resulted in suppressed cytotoxic activity of T cells, indicating that CD2 is crucial to the cellular immune response against EBV-infected cells. To explore the mechanism of CD2 downregulation, CD2 transcript of the patient's blood cells was examined by RT-PCR. The result barely demonstrated the CD2 transcript of normal length, suggesting that CD2 downregulation was caused by its decreased transcriptional activity and not by the production of abnormal transcript. CD2 gene is comprised of 5 exons, and the promoter regions were previously identified in 5′- and 3'-flanking regions. Although no mutation was found within the exons or known 5'- and 3'-promoter regions of CD2 gene of the patient's blood cells, 1 homozygous mutation was detected in intron 4. In addition, 2 homozygous single nucleotide polymorphisms (SNPs) were observed in exon 5 and 3'-flanking region of CD2, which suggested LOH of CD2 allele. The mutation found in intron 4 was suspected to impair an AP-1 binding motif, and luciferase reporter assay using Jurkat cell line demonstrated that this mutation led to significant decrease in transcriptional activity of the region. This is the first report that has indicated LOH of the mutant CD2 allele is one of the underlying mechanisms of ICL. Our findings suggest that some cases of unexplained immunodeficiency may be caused by genetic abnormalities of immunoregulatory genes in apparently normal, non-malignant hematological cells. Disclosures:No relevant conflicts of interest to declare.

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Abstract LB-20: Transcriptionally inactive PAX3-FKHR fails to induce ARMS cells differentiation

Abstract Alveolar rhabdomyosarcoma (ARMS) is an aggressive muscle cancer of childhood and believed to arise from committed muscle progenitor cells that have arrested during the process of skeletal muscle differentiation. One of the most established genetic signatures in ARMS is the presence of chimeric aberrant transcription factor PAX3-FKHR generated by fusion of PAX3 and FKHR genes. The stronger transcriptional activity of this fusion PAX3-FKHR protein has previously been attributed to the deregulation of genes, which are known to be associated with tumorigenesis and suppression of skeletal muscle differentiation. Currently, we are investigating the relative contribution of PAX3-FKHR's transcriptional activity in abrogating ARMS cells differentiation using mouse ARMS cells as an in vitro model differentiation readout system. Here, we show that transactivation potential of PAX3-FKHR is declined in mouse ARMS cells that have been induced to differentiate by growing these cells in mitogens-deficient in vitro differentiation-permissible condition. Moreover, this diminished activity of PAX3-FKHR is not due to the alteration of its sub-cellular localization or changes in the level of either mRNA or protein in these ARMS cells. Results also show the lack of muscle-specific gene activation in these ARMS cells despite the reduced transcriptional activity of PAX3-FKHR under condition of differentiation. We also observed that decrease in transcriptional activity of PAX3-FKHR is coincided with the activation of PI3/AKT pathway. The results further show that blockade of PI3/AKT pathway sustains the transcriptional activity of PAX3-FKHR whereas forced activation of AKT inhibits this activity. Together, our results signify that transcriptionally inactive PAX3-FKHR by AKT is incompetent to induce mouse ARMS cells differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-20.

Global Gene Silencing is Caused by the Dissociation of RNA Polymerase II from DNA in Mouse Oocytes

As mouse oocytes approach maturity, a global repression of gene transcription occurs. Here, we investigated the involvement of RPB1, the largest subunit of RNA polymerase II (RNAP II), in the regulation of this transcriptional silencing mechanism. Using BrUTP to follow transcription in an in vitro run-on assay, we observed an abrupt decrease in transcriptional activity when oocytes reached their full size (approximately 80 µm). Immunoblotting using antibodies specific for the phosphorylated and unphosphorylated forms of RPB1 revealed that RPB1 is phosphorylated at Ser-2 and Ser-5 in the small growing oocytes in which active transcription occurs. By contrast, in transcriptionally inactive, full-grown oocytes, RPB1 is predominantly unphosphorylated. When we permeabilized the nuclear membrane using Triton X-100 during fixation for immunocytochemistry, the unphosphorylated form of RPB1 diffused out of the nucleus in the full-grown oocytes but still remained there in the small growing oocytes, indicating that RPB1 is not bound to DNA in full-grown oocytes. These results suggest that the immediate cause of global transcriptional silencing is the dissociation of RNAP II from the DNA. We also observed dissociation of RPB1 from the DNA in full-grown oocytes treated with trichostatin A to decondense their chromatin, suggesting that chromatin condensation is not an essential process in gene silencing during oocyte growth.

A Missense GATA3 Mutation, Thr272Ile, Causes the Hypoparathyroidism, Deafness, and Renal Dysplasia Syndrome

The hypoparathyroidism, deafness, renal dysplasia (HDR) syndrome is caused by mutations in the gene encoding GATA3, which belongs to a family of dual zinc-finger transcription factors that have a role in vertebrate embryonic development. The aim of the study was to identify the GATA3 mutation in a HDR patient and determine its functional consequences. PATIENT AND DESIGN: A patient with HDR was studied after approval from the local ethical committee. Leukocyte DNA was used with GATA3-specific primers for PCR amplification, and the DNA sequences of the PCR products were determined. Wild-type and mutant GATA3 constructs were transfected into COS-7 cell, and their functions were assessed by Western blot analysis, immunocytochemistry, EMSAs, luciferase reporter assays, and three-dimensional modeling. A novel missense mutation, Thr272Ile, in zinc finger 1 (ZnF1) of GATA3 was identified. Western blot analysis and immunofluorescence revealed that the mutation did not affect nuclear localization of GATA3. However, EMSAs showed it to reduce DNA binding affinity, but not stability, and yeast two-hybrid assays demonstrated that the mutant GATA3 resulted in a loss of interaction with ZnF1 and ZnF6 of the cofactor FOG2. The mutant GATA3 significantly reduced luciferase reporter activity by more than 65% (P < 0.001), and three-dimensional modeling indicated that the functional abnormalities may be due to a loss of Thr272 polar side chain interaction with Leu268. A novel missense HDR-associated GATA3 mutation, Thr272Ile, has been identified and shown to result in reduced DNA binding, a partial loss of FOG2 interaction, and a decrease in gene transcription.

The role of the rat sarcoplasmic reticulum Ca2+-ATPase promoter in myocardial ischemia-preconditioning

Intracellular calcium ion concentration ([Ca2+]i) is a key regulator of ischemia-reperfusion injury (IRI) and ischemic preconditioning (IPC), with myocardial sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) acting as the major regulatory protein in the control of [Ca2+]i. In this study, we examined the effect of the SERCA2a promoter in IRI and IPC. The SERCA2a promoter fragment was acquired using PCR and molecular cloning technology, and was inserted into a luciferase reporter gene vector to construct reporter gene plasmids. Primary cultured neonatal rat cardiomyocytes were treated by analog ischemic treatment to produce in vitro models of IRI and IPC. Using an in vitro gene transfer assay, SERCA2a transcriptional activity was measured under conditions of IRI and IPC. Results from this study showed that (1) short duration analog ischemia treatment (analog IPC) results in a significant decrease in transcriptional activity, (2) analog IRI caused a dramatic depression in SERCA2a gene expression in transcription initiation levels, and (3) analog IPC attenuated IRI-induced transcriptional depression, even though IPC itself significantly depressed transcriptional activity. In conclusion, the SERCA2a promoter plays a significant role in IRI and IPC, with IPC able to precondition the SERCA2a promoter against the deleterious effects of IRI-induced injury.

Evaluation of transcriptional activity of p53 in individual living mammalian cells

To estimate the transcriptional activity of p53 in individual living mammalian cells, we constructed the enhanced green fluorescent protein–red fluorescent protein (EGFP–DsRed) reporter system with the EGFP–p53 expression vector and the reporter plasmid, which carried a p53-dependent promoter. The expression level and transcriptional activity of EGFP–p53 were determined simultaneously by green and red fluorescence signals, respectively. In this system, we could target only the cells expressing p53 at endogenous levels, as observed in UV- or adriamycin-stimulated A549 cells. Using this system, we investigated the transcriptional activity of mutant p53s in tetramerization domain. Transcriptional activities were nearly abolished by seven mutations and significantly reduced in several mutant p53s. However, under overexpression conditions, the latter mutant p53s showed activity similar to that observed in wild-type p53. These results indicated the importance of physiological concentration for p53 proteins in cells so as to analyze their activities. Fluorescence intensity distribution analysis indicated that the mutant p53s lacking transcriptional activity presented as monomer forms in the cellular extract. In most of the mutant p53s, the decrease in transcriptional activity correlated with an increase in the fraction of monomers. This reporter system can be used for estimating the transcriptional activity of mutant p53s without contribution of the cells overexpressing p53.

Association between NR2B subunit gene ( GRIN2B) promoter polymorphisms and sporadic Alzheimer’s disease in the North Chinese population

N-methyl- d-aspartate (NMDA) receptor plays a crucial role in learning, memory and information processing of human brain. Its dysfunction is related to the pathogenesis of Alzheimer’s disease (AD). We detected four polymorphisms of the promoter regions of the human NMDA receptor 2B (NR2B) subunit gene ( GRIN2B) in 362 AD patients and 334 healthy in North Han Chinese populations, these were −200T/G (rs1019385), −421C/A (rs3764028), −1447T/C (ENS10557853), and −1497G/A (rs12368476). Genetic analysis confirmed that there were significant differences in genotype ( P = 0.029) and allele ( P = 0.010) frequencies for −421C/A between SAD and control. In the subjects without APOE ɛ4 allele, these difference remained significant (genotype P = 0.012, allele P = 0.004). The −421CC genotype was about 1.5 fold increasing risk compared with CA + AA genotypes (OR = 1.517, 95% CI 1.077–2.137, P = 0.017). Luciferase reporter assay showed a 34.69–39.79% decrease in transcriptional activity for −421C allele of GRIN2B promoter compared with −421A in SH-SY5Y and HeLa cell lines. Our study suggests that the −421C allele may induce lower GRIN2B transcriptional activity and NR2B protein expression, thus it is associated with AD.

Transcriptional regulation of GSDML gene by antisense-oriented HERV-H LTR element

During the course of hominoid evolution, a new transcript variant of the GSDML (gasdermin-like protein) gene was formed by the integration of the antisense-oriented HERV-H (human endogenous retrovirus) LTR (long terminal repeat) element. To investigate regions that are critical for transcriptional regulation of the GSDML gene, we generated seven deletion mutants from a full-length clone (clone 1/630) that includes the HERV-H LTR sequence and compared their expression levels relative to the full-length parental clone using a transient transfection assay. In the transient transfection assay, deletion of the 5' flanking region (cellular origin) of the HERV-H LTR sequence led to a 4.5-fold increase in expression compared to the full-length clone, while deletion of the U5 region showed a significant decrease in transcriptional activity. Deletion of the 3' flanking region of the LTR sequence (clone 42/451) showed similar transcriptional activity to a clone missing the 5' flanking region of cellular origin (clone 42/630). Taken together, these data indicate that the HERV-H LTR sequence (viral origin) positively regulates transcriptional activity of the GSDML gene and that the 5' flanking region sequence (cellular origin) exerts negative transcriptional regulation.

Cooperative regulation of p73 promoter by Yin Yang 1 and E2F1

The transcriptional factor Yin Yang 1 (YY1) plays multifunctional role in various biological processes. Here we report that the silencing of YY1 using siRNA expression vectors in U2OS cells led to a significant decrease in transcriptional activity of p73, which is a member of p53 family. Consistently, overexpression of YY1 could enhance the transcriptional activity of p73 in a dose-dependent manner. Moreover, synergistic cooperation between YY1 and E2F1, through a mechanism involving a physical interaction, was observed in the regulation of p73 promoter. In conclusion, our results provide new insights into the function of YY1, and might clarify the complex regulation mechanism by YY1 network in diverse biological processed, such as development, differentiation and cancer biology.

Aspirin Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Hormone-Refractory Prostate Cancer Cells through Survivin Down-Regulation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether acetylsalicylic acid (ASA), so-called aspirin, enhances TRAIL-induced apoptosis in androgen-dependent LNCaP and androgen-independent LNCaP-derived prostate cancer cells. To evaluate the cell death effects of TRAIL in combination with ASA on tumor cells, we performed DNA fragmentation assay and immunoblot analysis for poly(ADP-ribose) polymerase-1, caspases, and anti-apoptotic proteins. We observed that ASA promoted TRAIL-induced apoptotic death in both LNCaP and its derived cells (C4, C4-2, and C4-2B). These enhancements of TRAIL's effect were related to the decrease in survivin protein expression by pretreatment with ASA. We also confirmed that knockdown in survivin expression by transfecting survivin small interfering RNA increased TRAIL-induced apoptosis. To study the mechanism of survivin down-regulation, we determined the levels of mRNA and the activities of survivin promoter in the ASA-treated and untreated cells. Reduction of the intracellular levels of survivin protein was due to a decrease in transcriptional activity. Data from electrophoretic mobility shift assay and chromatin immunoprecipitation analyses revealed that ASA inhibited the transcription factor E2F-1 binding activity to the survivin promoter region, which is known to regulate survivin gene transcription. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated by down-regulating survivin, and the down-regulation of survivin is due to inhibition of E2F-1 binding activity to the survivin promoter region.