GC Box Research Articles

ZNF32 protects against oxidative stress-induced apoptosis by modulating C1QBP transcription.

Reactive oxygen species (ROS)-driven oxidative stress has been recognized as a critical inducer of cancer cell death in response to therapeutic agents. Our previous studies have demonstrated that zinc finger protein (ZNF)32 is key to cell survival upon oxidant stimulation. However, the mechanisms by which ZNF32 mediates cell death remain unclear. Here, we show that at moderate levels of ROS, Sp1 directly binds to two GC boxes within the ZNF32 promoter to activate ZNF32 transcription. Alternatively, at cytotoxic ROS concentrations, ZNF32 expression is repressed due to decreased binding activity of Sp1. ZNF32 overexpression maintains mitochondrial membrane potential and enhances the antioxidant capacity of cells to detoxify ROS, and these effects promote cell survival upon pro-oxidant agent treatment. Alternatively, ZNF32-deficient cells are more sensitive and vulnerable to oxidative stress-induced cell injury. Mechanistically, we demonstrate that complement 1q-binding protein (C1QBP) is a direct target gene of ZNF32 that inactivates the p38 MAPK pathway, thereby exerting the protective effects of ZNF32 on oxidative stress-induced apoptosis. Taken together, our findings indicate a novel mechanism by which the Sp1-ZNF32-C1QBP axis protects against oxidative stress and implicate a promising strategy that ZNF32 inhibition combined with pro-oxidant anticancer agents for hepatocellular carcinoma treatment.

The Role of the Y Box Binding Protein 1 C-Terminal Domain in Vascular Endothelial Cell Proliferation, Apoptosis, and Angiogenesis

Different domains of the multifunctional transcription factor Y-box binding protein 1 (YB1) regulate proliferation, differentiation, and apoptosis by transactivating or repressing the promoters of various genes. Here we report that the C-terminal domain of YB1 (YB1 CTD) is involved in endothelial cell proliferation, apoptosis, and tube formation. The oligo pull-down assays demonstrated that YB1 directly binds double-stranded GC box sequences in endothelial cells through the 125-220 amino acids. Adenovirus expression vectors harboring green fluorescent protein (GFP) or GFP-tagged YB1 CTD were constructed and used to infect EA.hy926 endothelial cells. Overexpression of the YB1 CTD significantly increased p21 expression, decreased cyclin B1 expression, and inhibited the proliferation of EA.hy926 cells. YB1 CTD overexpression also increased Bax and active caspase 3 expression, decreased Bcl-2 expression, and induced apoptosis in EA.hy926 cells. Furthermore, overexpression of the YB1 CTD significantly suppressed migration and tube formation in EA.hy926 cells. Finally, YB1 CTD decreased ERK1/2 phosphorylation in EA.hy926 cells. These findings demonstrated vital roles for YB1 in endothelial cell proliferation, apoptosis, and tube formation through transcriptional regulation of GC box-related genes.

TALENs-Assisted Multiplex Editing for Accelerated Genome Evolution To Improve Yeast Phenotypes.

Genome editing is an important tool for building novel genotypes with a desired phenotype. However, the fundamental challenge is to rapidly generate desired alterations on a genome-wide scale. Here, we report TALENs (transcription activator-like effector nucleases)-assisted multiplex editing (TAME), based on the interaction of designed TALENs with the DNA sequences between the critical TATA and GC boxes, for generating multiple targeted genomic modifications. Through iterative cycles of TAME to induce abundant semirational indels coupled with efficient screening using a reporter, the targeted fluorescent trait can be continuously and rapidly improved by accumulating multiplex beneficial genetic modifications in the evolving yeast genome. To further evaluate its efficiency, we also demonstrate the application of TAME for significantly improving ethanol tolerance of yeast in a short amount of time. Therefore, TAME is a broadly generalizable platform for accelerated genome evolution to rapidly improve yeast phenotypes.

DNA methylation of the GC box in the promoter region mediates isolation rearing-induced suppression of srd5a1 transcription in the prefrontal cortex

The levels of allopregnanolone (ALLO), a neurosteroid, in brain and serum are related to severity of depression and anxiety. Steroid 5α-reductase type I is the rate-limiting enzyme in ALLO biosynthesis and plays an important role in control of the ALLO level in mammalian brain. In this study, we examined an epigenetic mechanism for transcriptional regulation of srd5a1, which codes for steroid 5α-reductase type I, using isolation-reared mice. The mRNA level of srd5a1 was decreased in the prefrontal cortex (PFC) in isolation-reared mice. Rearing in social isolation increased methylation of cytosines at −82 and −12bp downstream of the transcription start site, which are located in a GC box element in the promoter region of srd5a1. Binding of Sp1, a ubiquitous transcription factor, to the GC box was decreased in the promoter region of srd5a1 in the PFC in isolation-reared mice. Site-specific methylation at cytosine −12 of a srd5a1 promoter-luciferase reporter construct, but not that of cytosine −82, downregulated the promoter activity of srd5a1. These findings suggest that transcription of srd5a1 in brain is regulated by environmental factor-induced cytosine methylation in the promoter region. This finding could contribute to development of antidepressant and anxiolytic agents.

Efficient cleavage of DNA oligonucleotides by a non-FokI-type zinc finger nuclease containing one His₄-type finger domain derived from the first finger domain of Sp1.

In this study, we sought to improve the hydrolytic activity of a His4-type single finger domain (f2), which was previously derived from the second finger domain (f2') of the Sp1 zinc finger protein (Sp1wt), which has 3 tandem finger domains (f1', f2', and f3'). To this end, 2 His4-type single finger domains were generated by mutating 2 Cys residues participating in Zn(II) coordination with the His residues in the first (f1') and third finger (f3') domains of Sp1wt. Circular dichroism spectroscopy results showed that the first and second His4-type zinc finger domains (f1 and f2) adopted folded ββα structures in the presence of Zn(II), but that the third His4-type zinc finger domain (f3) did not. Non-FokI-type zinc finger nucleases containing 3 or 4 finger domains were also prepared by combining a His4-type zinc finger domain with the Sp1wt scaffold. We studied their DNA-binding abilities and hydrolytic activities against DNA oligonucleotides by performing gel-mobility-shift assays. The results showed that f1 had higher hydrolytic activity for a DNA oligonucleotide with a GC box (5'-GGG GCG GGG-3'), compared with that of f2, although both His4-type single finger domains had similar DNA-binding affinities. The difference in the hydrolytic activity between f1 and f2 was ascribed not only to the zinc coordinate structure, but also to its folding structure and the stability of finger domain.

Role of Specificity Protein 1, Hepatocyte Nuclear Factor 1α, and Pregnane X Receptor in the Basal and Rifampicin-Induced Transcriptional Regulation of Porcine Cytochrome P450 3A46.

Cytochrome P450 (CYP) 3A46, one of human CYP3A4 homologs, functions as a key enzyme in the metabolism of xenobiotics in pigs. However, the regulatory mechanism for the transcriptional activation of CYP3A46 in porcine liver remains unknown. In this study, we confirmed that CYP3A46 is constitutively expressed in porcine primary hepatocytes, and its expression was significantly induced by rifampicin (RIF) instead of dexamethasone. We further found that a proximal GC box and a distal hepatocyte nuclear factor 1 (HNF1) binding site within the 5'-flanking region of CYP3A46 are the important cis-regulatory elements involved in regulating the constitutive expression of CYP3A46, via recruiting specificity protein 1 (Sp1) and HNF1α, respectively. Furthermore, we revealed that HNF1α and pregnane X receptor (PXR) activate the RIF-mediated transcription of CYP3A46 by binding to the distal HNF1 binding site and the proximal direct repeats of AGGTCA separated by 4 bases motif, respectively. Meanwhile, HNF1α is also involved in regulating RIF-induced expression of CYP3A4 through a novel distal HNF1 binding site identified in the xenobiotic-responsive enhancer module. In summary, our data demonstrate that several transcription factors, including Sp1, HNF1α, and PXR, function in the basal and RIF-mediated transcriptional regulation of CYP3A46 by binding to their related cis-regulatory elements in the proximal promoter and distal enhancer.

Abstract 710: A Novel Mechanism by Which CPI-17 Is Down-regulated in Cultured Vascular Smooth Muscle Cells and Tissues by TNFα and in C57BL/6 Mice by Lipopolysaccharide

Objectives: Sepsis is characterized by a severe inflammatory response to infection, and its complications, including hypotension, can be fatal. It has long believed that hypotension in sepsis occur as a result of failure of the vascular smooth muscle cells (VSMC) to constrict. However, the molecular mechanism that links inflammation and decreases in vasoconstriction is largely unknown. Approaches and Results: CPI-17, a key regulator in vasoconstriction, was markedly decreased in mesentery arteries (MA) in mice injected with lipopolysaccharide (LPS). Incubation of cultured MA or VSMC with TNFα had similar effects on CPI-17 as LPS injection in mice. To identify transcriptional factors that respond to TNFα to suppress CPI-17 expression, we cloned a 1 kb murine CPI-17 promoter (-1,015 to +2 bp). Promoter deletion analysis revealed that a CPI-17 promoter region (-115 to -50 bp) was critical for TNFα to inhibit CPI-17 promoter activity. Bioinformatics analysis revealed this region contained 3 GC boxes that are putative binding sites for transcription factors SP1 and KLF4. Mutation of the 2nd and 3rd GC boxes, but not 1st GC box, abolished TNFα-mediated inhibitory effect on CPI-17 promoter activity. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) illustrated that SP1 and KLF4 bound to the 2nd and 3rd GC boxes, but not 1st GC box. Interestingly, TNFα had no effect on SP1 expression but suppressed SP1 binding to the CPI-17 promoter. In contrast, TNFα increased KLF4 expression and KLF4 binding to the CPI-17 promoter. Down-regulation of either SP1 or KLF4 by siRNA abolished inhibition of CPI-17 promoter activity by TNFα but the effects were opposite: SP1 siRNA decreased CPI-17 promoter activity whereas KLF4 siRNA increased CPI-17 promoter activity. Moreover, KLF4 up-regulation, but not SP1, was also found in MA in mice injected with LPS. KLF4 up-regulation by LPS preceded CPI-17 down-regulation and correlated with a decrease in vasoconstriction and hypotension. Conclusions: These studies reveal a novel mechanism by which CPI-17 is down-regulated by LPS/TNFα through SP1 and KLF4 in vascular wall and identify SP1, KLF4, and CPI-17 as new potential therapeutic targets for treatment of hypotension in patients with sepsis.

Gata3 cooperates with Gcm2 and MafB to activate parathyroid hormone gene expression by interacting with SP1

Haploinsufficiency of the Gata3 gene, which encodes a zinc-finger transcription factor, is associated with the disorder hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome in humans. However, the roles of Gata3 in transcriptional regulation in the parathyroid glands are not well-understood. In this study, we show that Gata3 activates transcription of parathyroid hormone (PTH), which is secreted from parathyroid glands and is critical for regulating serum calcium and phosphate homeostasis. Gata3 interacted with Gcm2 and MafB, two known transcriptional regulators of parathyroid development, and synergistically stimulated the PTH promoter. An SP1-binding element (GC box) located within the PTH-promoter proximal region was critical for activating transcription by Gata3. In addition, the ubiquitous transcription factor SP1 also interacted with Gata3 as well as MafB and Gcm2, and HDR syndrome-associated Gata3 mutants were defective in activating the PTH promoter. These results suggest that Gata3 is a critical regulator of PTH gene expression.

Identification and analysis of the promoter region of the human HAS3 gene

Hyaluronan (HA) is a key component of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the hyaluronan synthases including HAS1, HAS2 and HAS3. The expression and regulation of HAS1-3 are implicated in numerous physiological and pathological processes. The promoters of human HAS1 and HAS2 genes have been identified previously whereas HAS3 promoter remains unclear. In the present study, we have for the first time identified and characterized the human HAS3 gene promoter region. 5′ RACE assay revealed two novel transcriptional variants of HAS3 gene with distinct transcription start sites. Progressive deletion analysis of the 5′-flanking region of HAS3 gene demonstrated that HAS3 proximal promoter is mainly restricted to a 450-bp region (i.e. −761 to −305 bp upstream of the major HAS3 transcription start site), whereas its core promoter is located to a minimal 129-bp region (i.e. −433 to −305 bp upstream of the major HAS3 transcription start site). Transcriptional factor binding analysis indicated that HAS3 gene promoter lacks of canonical TATA box, but contains classical GC box as well as other putative binding sites for transcriptional factors such as C/EBP and NFκB. In addition, site-directed mutagenesis assay demonstrated that the proximal Sp1 binding site is essential for the robust proximal promoter activity of HAS3 gene whereas the core MTE (core promoter motif ten elements) motif is required for the basic core promoter activity of HAS3 gene. Our present study should facilitate further studies on the mechanism regulating the expression of this important gene.

Zinc finger independent genome-wide binding of Sp2 potentiates recruitment of histone-fold protein Nf-y distinguishing it from Sp1 and Sp3.

Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The Specificity proteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs in vitro. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions in vivo raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters in vivo. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin in vivo.

Cloning and functional analysis of Δ6-desaturase gene and its upstream region from Mortierella sp. AGED.

Δ6-desaturase belonging to membrane-bound enzyme is a key enzyme involved in the synthesis of polyunsaturated fatty acids (PUFAs). This study aimed to clone and characterise Δ6-desaturase gene and its upstream regulatory region of Mortierella sp. AGED. Glucose and soybean meal are best for lipid and arachidonic acid accumulation of Mortierella sp. AGED. A 1375-bp Δ6-desaturase gene AGfad6 which contains a 1275-bp open reading frame encoding 424 amino acids without signal peptide was cloned. The putative protein contained three conserved histidine-rich motifs and a conserved cytochrome b5 HPGG (H: Histine, P: Proline, G: Glycine, G: Glycine) motif, with a mass of 48.3 kDa and an isoelectric point of 5.96. AGfad6 was successfully expressed in Pichia pastoris GS115, which exerted the effect on converting linoleic acid to γ-linolenic acid. The 1712-bp upstream region contained basic transcriptional elements including TATA, GC and GATA box, putative target-binding sites for transcription factors such as TATA binding protein, transcription activator, CCAAT-enhancer-binding protein, activator protein 1, alcohol dehydrogenase gene regulator 1 and metabolic regulators p40x in fungi, stress-related elements including GT-1 (light-responsive, salicylic acid-inducible), stress response element, heat stress-responsive element, which might participate in regulation of PUFAs synthesis. The present finding could enable us to understand the evolution and regulatory mechanism of Δ6-desaturase gene.

ABCB1 Expression in Acute Myeloid Leukemia (AML): A Possible Predictive Value for Treatment Resistance?

Introduction: Over the last years, there has been a tremendous increase in understanding acute myeloid leukemia (AML) biology and a great effort has been taken in order to improve AML chemotherapy strategies. However, the growing knowledge of leukemia associated molecular mechanisms just started to translate into improved outcome. With regard to conventional chemotherapy multidrug resistance (MDR) is a persisting problem and the impact of ABCB1 (MDR1) expression is still controversially discussed.Methods: In this study we evaluated the ABCB1 expression using qRT-PCR and gene expression profiling (Affymetrix U133plus2.0 arrays) in 250 diagnostic AML samples derived from patients enrolled on a prospective treatment trial of the German-Austrian AML Study Group (AMLSG 07-04 trial; NCT00151242), in which patients were treated with an intensive anthracycline/cytarabine-based induction therapy. Findings were also evaluated in 154 TCGA AML cases receiving a 7+3 induction treatment (data available at http://cancergenome.nih.gov/) and put into perspective with previous reports. Furthermore, we investigated ABCB1 expression associated gene signatures and examined epigenetic regulation mechanisms by COBRA and methyl-CpG immunoprecipitation sequencing (MCIp-seq) in selected cases.Results: Our global analysis showed that patients who obtained a complete response (CR) following double induction therapy had lower ABCB1 mRNA levels compared to patients with refractory disease (RD) (p=0.07). Regarding cytogenetic AML subtypes, ABCB1 mRNA levels varied among the different cytogenetic groups with the complex karyotype group showing the highest ABCB1 and the inv(16) group the lowest ABCB1 expression levels. A comparison of CR versus RD cases within the cytogenetically determined prognostic groups showed that in the intermediate [CN-AML, t(11q23), and other intermediate risk cytogenetic aberrations (othersinter)] and poor risk groups (complex karyotype and othershigh), RD patients presented with significantly higher ABCB1 mRNA levels (p=0.02). Similarly, patients with favorable risk cytogenetics [t(8;21) and inv(16)], who achieved a CR, presented with lower ABCB1 levels compared to the ones, who were refractory. Patients with the lowest ABCB1 expression quartile (ABCB1low) showed significantly longer event-free survival (EFS) times than patients in the highest quartile cohort (ABCB1high) (median EFS 322 vs 105 days; p=0.02), while no differences were observed with regard to overall survival. In accordance, there was a significant enrichment of RD cases in the ABCB1high patient group (p=0.03). Next, in order to better understand the regulation of ABCB1 in AML, we specifically evaluated the DNA methylation level of a previously identified GC box important for ABCB1 expression regulation in CML and we performed global analyses of the entire ABCB1 5′ region. While both analyses did not reveal significant differences, further investigation of an ABCB1 associated gene pattern showed a correlation with CD34 and KIT expression (p<0.001). This suggests that like in CML, ABCB1 might be regulated by WNT, and in line, normal CD34+ hematopoietic stem cells also showed high ABCB1 expression levels.Conclusions: In summary, our data provide further evidence for a potential impact of ABCB1 deregulation on the response to AML chemotherapy, especially in more stem cell like leukemia cohorts as well as cytogenetically high risk AML. While we are currently further investigating the involvement of the Wnt/β-catenin pathway in the regulation of ABCB1 transcription in AML, further integration of molecular findings are warranted to better decipher the underlying drug resistance mechanisms. Ultimately, these analyses will improve patient management by adding valuable predictive biomarkers. DisclosuresNo relevant conflicts of interest to declare.

Histone Hyperacetylation Up-regulates Protein Kinase Cδ in Dopaminergic Neurons to Induce Cell Death: RELEVANCE TO EPIGENETIC MECHANISMS OF NEURODEGENERATION IN PARKINSON DISEASE

The oxidative stress-sensitive protein kinase Cδ (PKCδ) has been implicated in dopaminergic neuronal cell death. However, little is known about the epigenetic mechanisms regulating PKCδ expression in neurons. Here, we report a novel mechanism by which the PKCδ gene can be regulated by histone acetylation. Treatment with histone deacetylase (HDAC) inhibitor sodium butyrate (NaBu) induced PKCδ expression in cultured neurons, brain slices, and animal models. Several other HDAC inhibitors also mimicked NaBu. The chromatin immunoprecipitation analysis revealed that hyperacetylation of histone H4 by NaBu is associated with the PKCδ promoter. Deletion analysis of the PKCδ promoter mapped the NaBu-responsive element to an 81-bp minimal promoter region. Detailed mutagenesis studies within this region revealed that four GC boxes conferred hyperacetylation-induced PKCδ promoter activation. Cotransfection experiments and Sp inhibitor studies demonstrated that Sp1, Sp3, and Sp4 regulated NaBu-induced PKCδ up-regulation. However, NaBu did not alter the DNA binding activities of Sp proteins or their expression. Interestingly, a one-hybrid analysis revealed that NaBu enhanced transcriptional activity of Sp1/Sp3. Overexpression of the p300/cAMP-response element-binding protein-binding protein (CBP) potentiated the NaBu-mediated transactivation potential of Sp1/Sp3, but expressing several HDACs attenuated this effect, suggesting that p300/CBP and HDACs act as coactivators or corepressors in histone acetylation-induced PKCδ up-regulation. Finally, using genetic and pharmacological approaches, we showed that NaBu up-regulation of PKCδ sensitizes neurons to cell death in a human dopaminergic cell model and brain slice cultures. Together, these results indicate that histone acetylation regulates PKCδ expression to augment nigrostriatal dopaminergic cell death, which could contribute to the progressive neuropathogenesis of Parkinson disease.

Abstract 3505: Regulation of Cyclin A1 promoter by the Wilms tumor gene, WT1

Abstract Cyclin A1(CCNA1) is a unique type of Cyclin A expressed predominantly in highly proliferative tissues such as the testis and bone marrow. In hematopoietic cells CCNA1 expression has been observed in CD34+ progenitor cells, leukemia stem cells and leukemia cell lines. Similarly, Wilms tumor 1 (WT1) gene expression has been observed in these highly proliferative, undifferentiated cells. Regulation of CCNA1 expression is poorly understood, but the CCNA1 gene promoter is a highly GC-rich TATA-less sequence, containing three potential WT1 binding sites. Chromatin immunoprecipitation analysis of two of the three sites revealed binding of the WT1 protein to chromatin in K562 leukemia cells. To assess the role of WT1 in CCNA1 regulation, activation of the CCNA1 promoter by WT1 was measured using luciferase reporter constructs in K562 cells. The distal construct (-1180/+145 ) contains two potential WT1 sites, and the activity of this reporter construct was up-regulated by WT1 co-transfection. In contrast, the proximal (-454/+145) promoter region contains no canonical WT1 binding sites. Suprisingly, the proximal promoter was also up-regulated by WT1 co-transfection. Transcription factor binding site analysis revealed 6 GC boxes -within the core promoter region of the proximal promoter construct that we predicted could serve as non-canonical WT1 binding sites. To determine the importance of these GC boxes for WT1 binding, we treated K562 cells with Mithramycin A, which blocks binding at GC rich sequences. We found that Mithramycin A treatment decreased the activity of both the distal and proximal promoter constructs. Finally we demonstrated that WT1 mediated activation of CCNA1 promoter required an intact DNA binding domain, as a mutant WT1 expression construct lacking the zinc finger domain failed to activate any of the CCNA1 promoter constructs. Overall these results show that WT1 binding can up-regulate the CCNA1 promoter. This observation supports our previous finding that overexpressing WT1 in K562 cells increased CCNA1 mRNA. These data suggest that elevated WT1 expression could mediate increased CCNA1 expression in leukemia cells and thereby enhance proliferation. Overall, our results are consistent with the hypothesis that both WT1 and CCNA1 behave as oncogenes in leukemia. Citation Format: Sony Pandey, Mustafa Moazam, Steven J. Kuerbitz, Gail C. Fraizer. Regulation of Cyclin A1 promoter by the Wilms tumor gene, WT1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3505. doi:10.1158/1538-7445.AM2014-3505

Transcription factor KLF9 suppresses the growth of hepatocellular carcinoma cells in vivo and positively regulates p53 expression

Krüppel-like factor 9 (KLF9) is known to be a tumor suppressor gene in colorectal tumors and glioblastoma; however, the functional status and significance of KLF9 in hepatocellular carcinoma (HCC) is unclear. We report here that KLF9 is downregulated in HCC tissues. Restoration of KLF9 significantly inhibited growth and caused apoptosis in SK-Hep1 and HepG2 cells. We found that KLF9 positively regulated p53 levels by directly binding to GC boxes within the proximal region of the p53 promoter. Moreover, in the presence of cycloheximide, KLF9 significantly increased p53 stability in HCC cells. Remarkably, ectopic expression of KLF9 was sufficient to delay the onset of tumors and to promote regression of the established tumors in vivo, suggesting that KLF9 plays a critical role in HCC development and that pharmacological or genetic activation of KLF9 may have potential in the treatment of HCC.

Examination of the regulation of galectin-3 expression in cancer.

Galectin-3, a member of a β-galactoside-binding protein family, is involved in normal growth development as well as cancer progression and metastasis, but the detailed mechanisms of its functions or its transcriptional regulations are not well understood. Besides, several regulatory elements such as GC box, CRE motif, AP-1 site, and NF-κB sites, the promoter of galectin-3 gene (LGALS3) contains several CpG islands that can be methylated during tumorigenesis of prostate leading to the gene silencing. Here we describe protocols for identification of galectin-3 DNA methylation, suppression of DNA methyltransferases to reactivate galectin-3 expression, and development of methylation-specific polymerase chain reaction (MS-PCR) to assess galectin-3 expression in various biological specimens such as tissue, serum, and urine samples.

The promoter mutation c.-259C>T (-3438C>T) is not a common cause of non-syndromic hearing impairment in Austria.

The objective of this study was to investigate the relevance of routine assessment of c.-259C>T in the Austrian newborn screening program. Homozygous and compound heterozygous mutations in the coding region of the human gene encoding gap junction protein GJB2 (Connexin 26) cause up to 50 % of neonatal autosomal recessive non-syndromic hearing impairment identified in Caucasian newborn screening programs. More recently, a null mutation in the GC box of the GJB2 basal promoter c.-259C>T has been described which causes hearing impairment by completely suppressing GJB2 promoter activity. We determined the occurrence of c.-259C>T in cases of non-syndromic hearing impairment lacking known pathogenic alterations in GJB2 (n = 43), a non-syndromic hearing impaired patient group (n = 15) bearing the heterozygous GJB2 mutations c.35delG, c.[79G>A];[341A>G] (p. [V27I];[E114G]), c.109G>A (p.V37I), c.154G>C (p.V52L), c.262G>T (p.A88S), c.269T>C (p.L90P) and c.551G>C (p.R184P) and in a normal hearing group lacking alterations in GJB2 (n = 50). In the analyzed groups, no occurrence of c.-259C>T was found. The c.-259C>T mutation, previously described as -3438C>T, is not a common cause of non-syndromic hearing impairment alone or together with heterozygous pathogenic GJB2 mutations that are statistically overrepresented in non-syndromic hearing impaired patient groups. Screening of newborns for c.-259C>T is therefore unlikely to be commonly found in Austrian NSHI patients but could make a significant contribution to non-syndromic hearing impairment in other populations.

ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter

One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance.

A Fast Multiple Attractor Cellular Automata with Modified Clonal Classifier Promoter Region Prediction in Eukaryotes

DNA is a very important component in a cell, which is located in the nucleus. DNA contains lot of information. For DNA sequence to transcript and form RNA which copies the required information, we need a promoter. So promoter plays a vital role in DNA transcription. It is defined as “the sequence in the region of the upstream of the transcriptional start site (TSS)”. If we identify the promoter region we can extract information regarding gene expression patterns, cell specificity and development. So we propose a novel fast multiple attractor cellular automata (MACA) with modified Clonal classifier for promoter prediction in eukaryotes. We have used three important features like TATA box, GC box and CAAT box for developing this classifier. The proposed classifier is tested with datasets from Eukaryotic Promoter Database, EPDnew which is a collection of promoters of human, mouse, zebrafish and D.melanogaster. In training phase of the classifier 100% specificity was obtained. In testing phase 84.5% sensitivity and 92.7% specificity was achieved in an average. The time taken to predict the promoter region of length 252 in an average is .7 nano seconds.

Specificity protein 1 regulates topoisomerase IIβ expression in SH-SY5Y cells during neuronal differentiation.

Topoisomerase IIβ (top IIβ) is a nuclear enzyme with an essential role in neural development. The regulation of top IIβ gene expression during neural differentiation is poorly understood. Functional analysis of top IIβ gene structure displayed a GC box sequence in its transcription promoter, which binds the nuclear transcription factor specificity protein 1 (Sp1). Sp1 regulates gene expression via multiple mechanisms and is essential for early embryonic development. This study seeks to determine whether Sp1 regulates top IIβ gene expression during neuronal differentiation. For this purpose, human neuroblastoma SH-SY5Y cells were induced to neuronal differentiation in the presence of all-trans retinoic acid (RA) for 5 days. After incubation with 10 μM RA for 3-5 days, a majority of the cells exited the cell cycle to become postmitotic neurons, characterized by the presence of longer neurite outgrowths and expression of the neuronal marker microtubule-associated protein-2 (MAP2). Elevated Sp1 and top IIβ mRNA and protein levels were detected and found to be positively correlated with the differentiation stage. Chromatin immunoprecipitation assay demonstrated an increased recruitment of Sp1 to the top IIβ promoter after RA treatment. Mithramycin A, a compound that interferes with Sp1 binding to GC-rich DNA sequences, downregulated the expression of top IIβ, resulting in reduced expression of MAP2 and decreased neurite length compared with the control group. Our results indicate that Sp1 regulates top IIβ expression by binding to the GC box of the gene promoter during neuronal differentiation in SH-SY5Y cells.