Transcription Factor Binding Activity Research Articles

Abstract 4193: TNF-α dynamically modulates genome-wide cross-regulation of cRel, ΔNp63 and TAp73 promoter binding and gene expression in head and neck cancer: TP53 and NFκB ChIP-Seq in HNSCC

Abstract We previously showed that cRel, ΔNp63, and TAp73, members of NF-kappaB and TP53 transcription factor (TF) families, together regulate broad biological processes critical for inflammation and cancer survival. However, the underlying molecular mechanisms explaining the global cross-regulation of transcriptome and interactive signal network have not been revealed. Here, we demonstrate that inflammatory cytokine TNF-α induces a dynamic, genome-wide increase in cRel and ΔNp63 chromatin occupancy after replacing TAp73 binding activity, using Illumina Next Generation ChIP-sequencing and fitting the data into a dynamic Poisson distribution model integrated in MACS. Globally, significant binding peaks (p<10-5) were highly enriched in promoter regions (∼10%). These binding peaks further showed significant enrichment around transcription starting sites (±2000bp), and co-localized peaks for any of the two TFs were prevalent within a distance of ∼200 bp. De novo motif search using Gibbs Sampler indicated that cRel and ΔNp63 bound to p53 motifs while TAp73 binding switched from p53 to AP1 motifs when treated with TNF-α. Motifs from these TFs also co-localized within ∼200 bp. Binding activities of individual transcription factors were validated by ChIP-PCR and oliogo-ELISA based binding assays. In addition, TNF-α modulated 1050 differentially expressed genes detected by microarray, of which one-third exhibited binding activity for at least one TF, and 73 genes were identified with co-binding activities for the three TFs simultaneously. Ingenuity Systems Pathway Analysis (IPA) and Gene Ontology revealed that these genes are enriched in cancer-related transcription factors and signaling pathways. Our data suggest a novel mechanism of genome-wide replacement of TAp73 by cRel and ΔNp63, supporting cross-regulation and interactive network between NF-kappaB and TP53 families. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4193. doi:1538-7445.AM2012-4193

Activation of ATF4 mediates unwanted Mcl-1 accumulation by proteasome inhibition

AbstractMyeloid cell leukemia-1 (Mcl-1) protein is an anti-apoptotic Bcl-2 family protein that plays essential roles in multiple myeloma (MM) survival and drug resistance. In MM, it has been demonstrated that proteasome inhibition can trigger the accumulation of Mcl-1, which has been shown to confer MM cell resistance to bortezomib-induced lethality. However, the mechanisms involved in this unwanted Mcl-1 accumulation are still unclear. The aim of the present study was to determine whether the unwanted Mcl-1 accumulation could be induced by the unfolded protein response (UPR) and to elucidate the role of the endoplasmic reticulum stress response in regulating Mcl-1 expression. Using quantitative RT-PCR and Western blot, we found that the translation of activating transcription factor-4 (ATF4), an important effector of the UPR, was also greatly enhanced by proteasome inhibition. ChIP analysis further revealed that bortezomib stimulated binding of ATF4 to a regulatory site (at position −332 to −324) at the promoter of the Mcl-1 gene. Knocking down ATF4 was paralleled by down-regulation of Mcl-1 induction by bortezomib and significantly increased bortezomib-induced apoptosis. These data identify the UPR and, more specifically, its ATF4 branch as an important mechanism mediating up-regulation of Mcl-1 by proteasome inhibition.

18α-Glycyrrhetinic Acid Down-Regulates Expression of Type I and III Collagen via TGF-Β1/Smad Signaling Pathway in Human and Rat Hepatic Stellate Cells

Objective: To investigate the effects of 18α-glycyrrhetinic acid (18α-GA) on the expression of type I and III collagen in human and rat hepatic stellate cells (HSC) and to explore the role of TGF-β1/Smad signaling pathway involved.Methods: Following 18α-GA treatment, the cell viability and cell growth were detected to determine the optimal concentration of 18α-GA. The expressions of TGF-β1/Smad signaling-related genes including type I and III collagen in human and rat HSCs before and after 18α-GA treatment were measured by real time PCR. The expression of related proteins was verified by western blot assay. The phosphorylation level of Smad2 and Smad3 was detected by immunocytochemistry. The DNA binding activities of SP-1, AP-1 and NF-κB were measured by both EMSA and ArrayStar transcription factor activity assay.Results: 18α-GA could decrease the mRNA and protein expression of Smad3, type I and III collagen, increase the Smad7 expression in human and rat HSCs (P<0.05), and reduce phosphorylation level of Smad3 at 24 h and 48 h after treatment. The DNA binding activities of transcription factors were suppressed by 18α-GA in human and rat HSCs at 24 h, and the activities reduced in a time dependent manner with the lowest activities at 48 h, especially for SP-1.Conclusion: 18α-GA could inhibit the mRNA and protein expression of type I and III collagen in human and rat HSCs, which may be attributed to down-regulation of Smad3, up-regulation of Smad7, and inhibition of DNA binding activities of SP-1, AP-1 and NF-κB.

The basic helix loop helix transcription factor twist1 is a novel regulator of ATF4 in osteoblasts

Parathyroid hormone (PTH) is an essential regulator of endochondral bone formation and an important anabolic agent for the reversal of bone loss. PTH mediates its functions in part by regulating binding of the bone-related activating transcription factor 4 (ATF4) to the osteoblast-specific gene, osteocalcin. The basic helix-loop-helix (bHLH) factors Twist1 and Twist2 also regulate osteocalcin transcription in part through the interaction of the C-terminal "box" domain in these factors and Runx2. In this study, we discovered a novel function of PTH: its ability to dramatically decrease Twist1 transcription. Since ATF4 is a major regulator of the PTH response in osteoblasts, we assessed the mutual regulation between these factors and determined that Twist proteins and ATF4 physically interact in a manner that affects ATF4 DNA binding function. We mapped the interaction domain of Twist proteins to the C-terminal "box" domain and of ATF4, to the N-terminus. Furthermore, we demonstrate that Twist1 overexpression in osteoblasts attenuates ATF4 binding to the osteocalcin promoter in response to PTH. This study thus identifies Twist proteins as novel inhibitory binding partners of ATF4 and explores the functional significance of this interaction.

Mechanistic insights into the link between visfatin gene C-1535T polymorphism and coronary artery disease: an in vitro study

Visfatin, a pro-inflammatory cytokine predominantly released from leucocytes, is correlated with coronary artery disease (CAD). We have previously reported that the -1535C>T polymorphism (rs1330082), which located on the promoter region of visfatin, was associated with decreased risk of CAD. Here, we investigated the underlying mechanism by which this polymorphism affects the genetic susceptibility to CAD. The difference of the promoter activities between -1535T variant and -1535C allele was tested by luciferase reporter gene assay. The difference of transcription factor binding activities between T and C allele was evaluated by electrophoretic mobility shift assay. In reporter gene assay, we showed that the T variant had a significantly reduced transcriptional activity compared with the C allele. The T-variant significantly attenuated the promoter binding affinity to nuclear transcription factors and this effect became much obvious after treatment with TNF-α. Moreover, competition experiment revealed that the retarded complex formed by T-1535- or C-1535-probe binding to nuclear extracts was nearly completely inhibited by unlabeled activator protein-1 (AP-1) specific probe, indicating that AP-1 might be the target nuclear effector. Taken together, our data provided potential mechanistic link between the visfatin -1535C>T polymorphism and reduced CAD risk.

S-Glutathionylation of Cysteine 99 in the APE1 Protein Impairs Abasic Endonuclease Activity

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a central participant in the base excision repair pathway, exhibiting AP endonuclease activity that incises the DNA backbone 5′ to an abasic site. Besides its prominent role as a DNA repair enzyme, APE1 was separately identified as a protein called redox effector factor 1, which is able to enhance the DNA binding activity of several transcription factors through a thiol-exchange-based reduction–oxidation mechanism. In the present study, we found that human APE1 is S-glutathionylated under conditions of oxidative stress both in the presence of glutathione in vitro and in cells. S-glutathionylated APE1 displayed significantly reduced AP endonuclease activity on abasic-site-containing oligonucleotide substrates, a result stemming from impaired DNA binding capacity. The combination of site-directed mutagenesis, biochemical assays, and mass spectrometric analysis identified Cys99 in human APE1 as the critical residue for the S-glutathionylation that leads to reduced AP endonuclease activity. This modification is reversible by reducing agents, which restore APE1 incision function. Our studies describe a novel posttranslational modification of APE1 that regulates the DNA repair function of the protein.

AhR deficiency impairs expression of LPS-induced inflammatory genes in mice

Recent reports suggest the participation of the aryl hydrocarbon receptor (AhR) in the induction mechanism of the NF-κB signaling pathway. In the current study we challenged C57BL/6 wild-type (WT) and AhR deficient (AhR−/−) mice with bacterial lipopolysaccharide (LPS) to investigate the role of the AhR in expression profiles of LPS and NF-κB target genes. Further, we analyzed the effect of LPS on the DNA binding activity of NF-κB, C/EBP and AP-1 transcription factors in liver and lung from WT and AhR−/− mice. The results show that the LPS-induced expression of several target genes was impaired in AhR−/− mice compared to WT mice. Depending on the target gene, the target tissue as well as the time of treatment, the deficiency of AhR may cause an inhibition or increase of the LPS-induced gene expression. The binding activity of NF-κB, C/EBP and AP-1 transcription factors was also affected in a time- and tissue-dependent manner. The current study shows that the AhR is implemented in LPS-induced inflammatory gene expression in vivo even in the absence of exogenous ligands of the AhR. The main implication of this finding is that the AhR functions in Toll-like receptor (TLR) and NF-κB signaling after activation by a classical stimulus, such as LPS.

Dual function of polycomb group proteins in differentiated murine T helper (CD4&lt;sup&gt;+&lt;/sup&gt;) cells

BackgroundFollowing antigen recognition, naive T helper (Th; CD4+) cells can differentiate toward one of several effector lineages such as Th1 and Th2; each expressing distinctive transcriptional profiles of cytokine genes. These cytokines eventually instruct the strategy of the immune response. In our search for factors that propagate the transcriptional programs of differentiated Th cells, we previously found that Polycomb group (PcG) proteins, which are known as epigenetic regulators that maintain repressive chromatin states, bind differentially the signature cytokine genes. Unexpectedly, their binding to the Ifng (Interferon-g) in Th1 cells and Il4 (Interleukin-4) in Th2 cells, was correlated with transcriptional activation. Therefore, in this study we aimed to determine the functional role of PcG proteins in the regulation of the expression of the signature cytokine genes.MethodsPcG proteins were knocked down in primary and established murine Th cells using transduction of lentiviruses encoding short hairpin RNAs (shRNAs) directed to Mel-18, Ezh2, Eed and Ring1A, representative of two different PcG complexes. The chromatin structure and the binding activity of PcG proteins and transcription factors at the Ifng promoter were assessed by chromatin immunoprecipitation (ChIP) assays.ResultsDownregulation of PcG proteins was consistent with their function as positive regulators of the signature cytokine genes in primary and established Th1 and Th2 cells. Moreover, the PcG protein Mel-18 was necessary to recruit the Th1-lineage specifying transcription factor T-bet, and the T cell receptor (TCR)-inducible transcription factor NFAT1 to the Ifng promoter in Th1 cells. Nevertheless, our results suggest that PcG proteins can function also as conventional transcriptional repressors in Th cells of their known target the Hoxa7 gene.ConclusionsOur data support a model whereby the non-differentially expressed PcG proteins are recruited in a Th-lineage specific manner to their target genes to enforce the maintenance of specific transcriptional programs as transcriptional repressors or activators. Although our results suggest a direct effect of PcG proteins in the regulation of cytokine gene expression, indirect functions cannot be excluded.

Endoplasmic reticulum stress induced by hepatitis B virus X protein enhances cyclo-oxygenase 2 expression via activating transcription factor 4

Chronic hepatitis B is a disease of the liver that can progress to cirrhosis and liver cancer. The HBx (hepatitis B virus X) protein of hepatitis B virus is a multifunctional regulator that induces ER (endoplasmic reticulum) stress by previously unknown mechanisms. ER stress plays a critical role in inflammatory induction and COX2 (cyclo-oxygenase 2) is an important mediator of this inflammation. In the present study, we demonstrate the molecular mechanisms of HBx on induction of ER stress and COX2 expression. In addition, HBx reduced expression of enzymes which are involved in mitochondrial β-oxidation of fatty acids and the mitochondrial inner membrane potential. The reduction in intracellular ATP levels by HBx induced the unfolded protein response and COX2 expression through the eIF2α (eukaryotic initiation factor 2α)/ATF4 (activating transcription factor 4) pathway. We confirmed that ATF4 binding to the COX2 promoter plays a critical role in HBx-mediated COX2 induction. The results of the present study suggest that HBV infection contributes to induction of hepatic inflammation through dysfunction of cellular organelles including the ER and mitochondria.

Bovine lactoferrin (LTF) gene promoter haplotypes have different basal transcriptional activities

Genetic polymorphisms present in the bovine lactoferrin (LTF) gene promoter have the potential to affect milk lactoferrin concentrations. The objectives were: (1) to identify, in silico, SNPs in the promoter region of the LTF gene that could affect transcription factor binding activity, (2) to investigate the effects of these SNPs in vitro by measuring promoter transcriptional activities of different bovine LTF promoter haplotypes and (3) to investigate the genetic association between LTF promoter SNPs and milk lactoferrin concentration. Haplotypes were deduced from sequencing of the 2.2-kb bovine LTF promoter in 78 unrelated animals. In silico analysis of the 2.2-kb promoter revealed two major haplotypes (BtLTF_H1a and BtLTF_H2a) that differed at 10 SNP loci that affect transcription factors of both a constitutive (at -28, -1702) and an inducible (at -131, -270, -586, -2047, -2077, -2122, -2140 and -2151) nature. The basal promoter transcriptional activity of BtLTF_H1a was 1.44-fold higher than that of BtLTF_H2a in mammary epithelial cells. Cows with the BtLTF_H1a haplotype had increased lactoferrin protein concentration in milk at various time points over the lactation curves, compared to herdmates with the BtLTF_H2a haplotype. The SNPs c.-28A>C, c.-131T>C, c.-156A>G, c.-270T>C, c.-586C>T, c.-1702A>G, c.-1953G>A, c.-2047A>G, c.-2077A>G, c.-2122C>T, c.-2140A>G and c.-2151G>A were associated (P < 0.001) with milk lactoferrin content in 372 Holstein-Friesian cows. The identification of bovine LTF promoter haplotypes with different basal transcriptional activities in vitro that are associated with lactoferrin levels in milk in vivo may facilitate the identification of designer dairy herds for increased lactoferrin content in milk.

Abstract PL01-04: ODC genotype as prognostic and predictive factors in colorectal carcinogenesis and prevention

Abstract Polyamines are increased in many intraepithelial neoplasia, which are risk factors for cancer in humans (1). Ornithine decarboxylase (ODC1), the first enzyme in polyamine synthesis, is regulated in part by Ebox transcription factors including MYC and MAD family members. The ODC1 gene contains several single nucleotide polymorphisms (SNPs) which are associated with risk of colorectal neoplasia. A SNP located 316 nucleotides from the start of transcription (rs2302615) has functional consequences for Ebox transcription factor binding and promoter activity. Both the transcription activator MYC and transcriptional repressors in the MAD family preferentially bind the ODC promoter element containing the minor A allele, which is flanked by two canonical E-boxes, resulting in allele-specific expression (2, 3). This SNP is associated with both risk of metachronous colorectal adenoma (CRA), in several independent studies (2, 4), and survival of patients with stages I-III colon cancer (3). The ODC1 SNP was also found to be predictive of reduced risk of CRA in association with aspirin use by three independent groups (2, 4, 5). The mechanism underlying the relationship between the ODC1 SNP and aspirin appears to involve the action of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) on polyamine catabolism and export. NSAIDs, including aspirin and sulindac, and celecoxib transcriptionally activate SAT1, a spermidine/spermine acetyltransferase, by unique mechanisms (6-8). Acetylation targets these polyamines for export by an SLC3A2-dependent arginine antiporter (9). Thus, NSAIDs and celecoxib, by activating polyamine export, complement ODC1 inhibitors, which suppress polyamine synthesis, to reduce tissue polyamine contents. In a prospective, randomized, placebo-controlled clinical trial of difluoromethylornithine (DFMO), a selective ODC1 inhibitor, and the NSAID sulindac for three years, combination treatment was associated with a 70% reduction of all, and over a 90% reduction of advanced and/or multiple CRAs, in patients with prior colon polyps (10). The 30% rate of non-advanced CRA development in the treatment arm was strongly associated with both the ODC1 SNP and dietary polyamines (11). Consideration of homeostatic regulatory processes, supported by measurements of tissue polyamine contents in participants in this trial, support the interpretation that decreased ODC transcription, resulting from the ODC1 SNP, is associated with altered polyamine transport. Treatment-associated toxicities were rare and associated with pretreatment clinical and genetic risk factors. The ODC1 SNP was predictive of ototoxicity in this trial (11). These studies identify genetic variability in ODC1, a MYC target gene, as a prognostic factor for colorectal carcinogenesis and as a predictive factor for treatments targeting this pathway. The role(s) of other ODC1 SNPs in colorectal or other cancers remains to be established.

A Functional Polymorphism In the CD95 Cell Death Receptor Associated with Prognosis In Acute Promyelocytic Leukemia

AbstractAbstract 756Treatment of acute promyelocytic leukemia (APL) has been revolutionized by the introduction of retinoid-based chemotherapy. However, up to 20% of patients fail to achieve or maintain remission. Understanding the mechanisms that underlie treatment failure remains an important goal. We genotyped a common G>A polymorphism at position -1377 (rs2234767) in the core promoter of the CD95 cell death receptor gene in 707 subjects with acute myeloid leukemia (AML) that included 233 patients with APL. We also tested the effect of the promoter variant on transcription factor binding and transcriptional activity. Compared to the GG genotype, carrier status for the A variant at position -1377 was significantly associated with worse prognosis in APL patients, but did not affect outcome in non-APL AML patients. In univariate analysis, APL patients who were GG homozygote were more likely to achieve complete remission (odds ratio (OR) 4.31, 95% confidence interval CI 1.44–12.9, p=0.009, Table) and had a better overall survival at 5 years (78% vs 64%, p=0.04) compared to heterozygotes and AA homozygotes. In multivariate Cox regression analysis that included performance status, WBC count, age, gender and secondary status in the model, carrier status for the CD95 -1377A allele (GA + AA) was a significant predictor of overall survival (OR 2.22 (1.23-4.02) p=0.007, Table) and was the strongest predictor of death within 8 weeks (OR 5.73 (2.26-14.49) p<0.0001, Table). Carriers were also significantly more likely to die from infection compared to GG homozygotes (21% vs 7%, OR 4.18, 95% CI 1.63–10.71, p=0.001). There was some evidence of an interaction between the CD95 variant and WBC count in that penetrance of the rare allele for poor outcome and infection-related death was stronger in patients with a higher WBC count at presentation. The A variant at position -1377 destroys a binding site for the stimulatory protein 1 (SP1) transcriptional regulator and is associated with lower transcriptional activity of CD95, demonstrating functional relevance of this common promoter variant. CD95 is a death receptor that regulates homeostasis in several tissues, including B and T lymphocytes. As such, differences in gene expression mediated by the -1377 variant could affect immune function. In conclusion, the G-1377A substitution represses CD95 expression by disrupting SP1 transcription factor binding and is significantly associated with poor prognosis and particularly early death in APL.Table 1Prognosis of APL cases stratified by CD95 -1377 genotype.OutcomeCD95-1377 status (Events/Patients (%))Unadjusted OR (95% CI); p valueAdjusted OR (95% CI); p valueGGGA + AAComplete Remission175/189 (93)35/44 (80)4.31 (1.44–12.9) p=0.0094.52 (1.48–13.86) p=0.005Death within 7 days5/189 (3)3/44 (7)3.46 (0.58–20.5) p=0.172.04 (0.35–11.91) p=0.4Death within 30 days11/189 (6)9/44 (20)6.16 (1.97–19.3) p=0.0025.00 (1.92–13.02) p=0.0003Death within 8 weeks11/189 (6)10/44 (23)7.45 (2.44–22.7) p=0.00045.73 (2.26–14.49) p<0.0001Overall survival (deaths)47/189 (25)17/44 (39)2.10 (1.09–4.08) p=0.042.22 (1.23–4.02) p=0.007Disease–free survival (relapse)43/175 (25)13/35 (37)1.82 (0.88–3.76) p=0.121.92 (0.98–3.59) p=0.06Death from infection13/189 (7)8/44 (18)4.68 (1.49–14.76) p=0.024.18 (1.63–10.71) p=0.001 Disclosures:No relevant conflicts of interest to declare.

Induction of intercellular adhesion molecule-1 by water-soluble components of Hericium erinaceum in human monocytes

Hericium erinaceum is a medicinal mushroom that has been traditionally used in Asian countries for the treatment of cancers and infectious diseases. Although the immunomodulating activity of H. erinaceum is considered to be responsible for its medicinal activity, its action mechanisms are poorly understood. In the present study, we investigated the capability of water-extracted H. erinaceum (WEHE) to induce the expression of intercellular adhesion molecule-1 (ICAM-1), which regulates the migration of immune cells. THP-1, a human monocytic cell-line, or human peripheral blood mononuclear cells (PBMC) were stimulated with WEHE (0-30 μg/mL) and subsequently analyzed using flow cytometry to examine the surface expression of ICAM-1 protein. Steady-state levels of ICAM-1 mRNA were estimated using real-time reverse transcription-polymerase chain reaction analysis. Electrophoretic mobility shift assay was conducted to examine transcription factors involved in ICAM-1 transcription. WEHE induced ICAM-1 expression at both protein and mRNA levels in THP-1 cells in a dose- and time-dependent fashion. A similar pattern of ICAM-1 induction was also observed in CD14(+) monocytes in human PBMC that were stimulated with WEHE. The ICAM-1 expression on THP-1 cells stimulated with WEHE was suppressed by specific inhibitors for extracellular signal-regulated kinases (ERK) and reactive oxygen species (ROS). Additionally, exposure of THP-1 cells to WEHE increased the DNA binding activities of NF-κB, AP-1, SP-1 and STAT-1 transcription factors, all of which are known to be required for ICAM-1 gene expression. These results suggest that WEHE induces ICAM-1 expression in human monocytes through ERK- and ROS-dependent signaling pathways, resulting in the subsequent activations of NF-κB, AP-1, SP-1, and STAT-1 transcription factors.

The effects of hepatitis C virus non-structural protein 3 on cell growth mediated by extracellular signal-related kinase cascades in human hepatocytes in vitro

Hepatitis C virus (HCV) infection has become a severe health problem worldwide. The viral proteins are believed to be among the most important factors that contribute to HCV mediated pathogenesis. Accumulated evidence demonstrating that HCV non-structural protein 3 (NS3) possesses oncogenic potential, and is involved in the regulation of cell proliferation has been documented. In this study, we emphasized the effect of HCV NS3 protein on cell proliferation in the immortally normal hepatocyte QSG7701 cells. The cell line transfected with plasmid expressing NS3 protein showed enhanced cell growth, extracellular signal-related kinase (ERK) activation, DNA binding activities of transcription factors of activator protein 1 (AP-1) and NF-kappaB, and cyclin D1 overexpression, but without activation of Jun amino-terminal kinase or p38. Pre-treatment of NS3 protein expressing cells with ERK inhibitor, PD98059, blocked the activation of AP-1 and NF-kappaB, and inhibited cyclin D1 expression and cell proliferation. The results suggest that NS3-mediated cell growth occurs through activation of ERK/AP-1 and NF-kappaB/cyclin D1 cascades.

Abstract 4977: Identification of transcriptional mechanisms specific to of NF1 gene deficiency

Abstract Neurofibomatosis type 1 (NF1) is an autosomal dominant disease (1:3000) resulting from the loss-of-function mutation of the NF1 gene that encodes neurofibromin. Although deregulation of Ras and mTOR has been reported, the role of neurofibromin in NF1 disease pathogenesis is not fully understood. In this study, we developed a systems approach to identify aberrant gene regulation and transcription factor (TF) binding activities associated with loss-of-function of neurofibromin. Two malignant peripheral nerve sheath tumor (MPNST) cell lines were used. STS26T (NF1+/+) has the wild type of NF1 gene and ST88-14(NF1−/−) that has a mutant NF1 gene. Specifically, TF binding activities and gene expression changes related to N-Ras, active ERK and neurofibromin deficiency were determined by using Protein/DNA arrays and gene expression arrays, respectively. Three comparison systems have been employed in STS26T (NF1+/+): 1) inducible oncogenic N-Ras expression, 2) MAPK pathway inhibition by U0126, 3) NF1 gene siRNA knockdown. Similarly, we applied three systems to ST88-14(NF1−/−): 1) N-Ras siRNA knockdown, 2) MAPK pathway inhibition by U0126, 3) normal human Schwann cells vs. ST88-14(NF1−/−). Based on these comparisons, the significant TF binding activity changes due to activation of N-Ras, MAPK and neurofibromin deficiency were for the first time revealed as critical effector pathways in NF1 disease. We identified many TF binding activity changes and their possible relationships to N-Ras, MAPK pathway and neurofibromin deficiency. For example, the activity changes for MEF3 and GATA1/2 only occurred when NF1 gene is siRNA knockdown in STS26T (NF1+/+). Thus, neurofibromin may regulate the pathway independent of MAPK and N-Ras. On the other hand, the binding activity of Pax3 increased only in U0126 treated ST88-14(NF1−/−) indicating that the MAPK pathway regulates its function directly. All these changes were verified by electrophoretic mobility shift assays. Gene expression arrays are being used to confirm target gene expression due to TF banding activity changes and comparative genome hybridization will used to exclude gene expression changes resulting from the gene amplification. A method to study transcription regulatory pathways based on the relationship between transcription factor binding profiles and gene expression profiles will be established and used to identify aberrant gene regulation uniquely related to N-Ras, MAPK and neurofibromin, respectively. This systematic analysis of NF1 deficiency will provide an understanding of the gene dysregulation in this disease, which can facilitate novel drug target discovery. At same time, new functions of neurofibromin other than its activity as a Ras-GAP are being revealed through these analyses. 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 4977.

Abstract 4080: Global changes in gene expression during phorbol ester-induced differentiation of human acute myeloid leukemia cells

Abstract This study seeks to elucidate the molecular mechanisms by which the acute myeloid leukemia cell line, HL-60, is induced to differentiate into macrophage-like cells following exposure to phorbol 12-myristate 13-acetate (PMA). Changes in gene expression over the time course of PMA treatment were measured by DNA microarray analysis and select genes shown to be significantly up- and down-regulated were validated by quantitative, real-time PCR. Functional studies of transcription factor binding activities were performed by the electrophoretic mobility shift assay (EMSA). Novel findings include the rapid and sustained induction of mRNA levels for a group of CC chemokine genes (CCL2, CCL3, CCL3L1, CCL4, CCL4L1 and CCL4L2). These genes displayed 10 to 50-fold expression level increases that were observed within 3 hours of treatment and were maintained through 6 hours post-induction. The anti-proliferative effects of these peptides are being examined on a variety of leukemia cell lines. Among the most highly induced early genes were others known to encode inflammatory response proteins (TNF, IL8, C3AR1). In addition, the transcription factor profile is dramatically altered early in the differentiation time course, with the predicted activation of AP-1 family members (JUNB, FOSL1) and the concomitant inhibition of NFKB (TNFAIP3, NFKBIA, NFKBID). Other transcription factor genes were transiently up regulated (MAFB, GATA2, ETS2) and c-MYC expression was significantly down regulated and remained low during the differentiation course. Pathway analysis was used to predict gene expression changes occurring later in the differentiation process and was tested by microarray analysis. Early events in the genetic re-programming of HL-60 cells in response to the differentiation inducer PMA includes a massive up-regulation of chemokine genes and alteration of the transcription factor profile. The implication of these gene expression changes will be discussed in the context of differentiation therapies for acute myeloid leukemia. 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 4080.

The Site Specific Demethylation in the 5′-Regulatory Area of NMDA Receptor 2B Subunit Gene Associated with CIE-Induced Up-Regulation of Transcription

BackgroundThe NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5′-regulatory area following CIE treatment.MethodsPrimary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5′-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity.ResultsAnalysis of individual CpG methylation sites within the NR2B 5′regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors.ConclusionsThese results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.

DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA contacts. We use this effect to establish the separate contributions of transcription factor binding and DNA dynamics to transcriptional activity. Our results argue against a purely ‘transcription factor-centric’ view of transcription initiation, suggesting that both DNA dynamics and transcription factor binding are necessary conditions for transcription initiation.

Combinatorial binding predicts spatio-temporal cis-regulatory activity

Development requires the establishment of precise patterns of gene expression, which are primarily controlled by transcription factors binding to cis-regulatory modules. Although transcription factor occupancy can now be identified at genome-wide scales, decoding this regulatory landscape remains a daunting challenge. Here we used a novel approach to predict spatio-temporal cis-regulatory activity based only on in vivo transcription factor binding and enhancer activity data. We generated a high-resolution atlas of cis-regulatory modules describing their temporal and combinatorial occupancy during Drosophila mesoderm development. The binding profiles of cis-regulatory modules with characterized expression were used to train support vector machines to predict five spatio-temporal expression patterns. In vivo transgenic reporter assays demonstrate the high accuracy of these predictions and reveal an unanticipated plasticity in transcription factor binding leading to similar expression. This data-driven approach does not require previous knowledge of transcription factor sequence affinity, function or expression, making it widely applicable.

Critical Role of Activating Transcription Factor 4 in the Anabolic Actions of Parathyroid Hormone in Bone

Parathyroid hormone (PTH) is a potent anabolic agent for the treatment of osteoporosis. However, its mechanism of action in osteoblast and bone is not well understood. In this study, we show that the anabolic actions of PTH in bone are severely impaired in both growing and adult ovariectomized mice lacking bone-related activating transcription factor 4 (ATF4). Our study demonstrates that ATF4 deficiency suppresses PTH-stimulated osteoblast proliferation and survival and abolishes PTH-induced osteoblast differentiation, which, together, compromise the anabolic response. We further demonstrate that the PTH-dependent increase in osteoblast differentiation is correlated with ATF4-dependent up-regulation of Osterix. This regulation involves interactions of ATF4 with a specific enhancer sequence in the Osterix promoter. Furthermore, actions of PTH on Osterix require this same element and are associated with increased binding of ATF4 to chromatin. Taken together these experiments establish a fundamental role for ATF4 in the anabolic actions of PTH on the skeleton.