Inhibition Of Transcription Factor Binding Research Articles

Two-way feedback between chromatin compaction and histone modification state explains Saccharomyces cerevisiae heterochromatin bistability

Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. However, a broader hypothesis suggests that chromatin compaction can be both a cause and a consequence of the locus histone modification state, with a tight bidirectional interaction underpinning bistable transcriptional states. To rigorously test this hypothesis, we developed a mathematical model for the dynamics of the HMR locus in Saccharomyces cerevisiae, that incorporates activating histone modifications, silencing proteins, and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states (and vice versa), and protein binding/histone modification levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. Such bidirectional feedback between chromatin compaction and the histone modification state may be a widespread and important regulatory mechanism given the hallmarks of many heterochromatic regions: physical chromatin compaction and dimerizing (or multivalent) silencing proteins.

Evidence that direct inhibition of transcription factor binding is the prevailing mode of gene and repeat repression by DNA methylation

Cytosine methylation efficiently silences CpG-rich regulatory regions of genes and repeats in mammalian genomes. To what extent this entails direct inhibition of transcription factor (TF) binding versus indirect inhibition via recruitment of methyl-CpG-binding domain (MBD) proteins is unclear. Here we show that combinatorial genetic deletions of all four proteins with functional MBDs in mouse embryonic stem cells, derived neurons or a human cell line do not reactivate genes or repeats with methylated promoters. These do, however, become activated by methylation-restricted TFs if DNA methylation is removed. We identify several causal TFs in neurons, including ONECUT1, which is methylation sensitive only at a motif variant. Rampantly upregulated retrotransposons in methylation-free neurons feature a CRE motif, which activates them in the absence of DNA methylation via methylation-sensitive binding of CREB1. Our study reveals methylation-sensitive TFs in vivo and argues that direct inhibition, rather than indirect repression by the tested MBD proteins, is the prevailing mechanism of methylation-mediated repression at regulatory regions and repeats.

Aberrant promoter hypermethylation regulates thrombomodulin in high altitude induced deep vein thrombosis

BackgroundDNA methylation regulates gene expression by inhibiting transcription factor binding to promoter and regulatory regions. Acute hypoxia during altitude exposure is associated with decreased natural anticoagulants and morbid thrombotic events. Thrombomodulin (TM) is a high affinity thrombin binding receptor protein, vital for vascular homeostasis. The purpose of this study is to determine gene expression regulation via methylation of TM gene in high altitude hypoxia induced deep vein thrombosis (DVT) patients. Materials and resultsPercent 5-methyl cytosine analysis showed increased methylation in high altitude DVT patients (HAP) as compared to high altitude control (HAC) and seal level control (Control) subjects, while TM protein and mRNA levels were decreased in high altitude DVT patients as compared to other two groups. Bisulfite sequencing analysis indicated increased methylation in TM promoter in high altitude DVT patients compared to high altitude controls. Flow cytometry analysis showed decreased TM expression in hypoxia induced primary human umbilical vein endothelial cells (HUVECs). Treatment with specific DNA methyltransferase (DNMT) inhibitor-decitabine during hypoxia, restored TM expression. in vitro global methylation assay showed increased methylation in hypoxia group. Specific concentration of decitabine in hypoxia decreased global methylation showing a direct correlation between DNMTs and methylation. Selective dose of decitabine restored TM levels in HUVECs. DNMT1 and DNMT3B proteins showed to mediate the overall expression of TM. ConclusionTM emerged as a potential candidate for methylation in high altitude DVT patients, regulated by hypoxia-induced epigenetic mechanism. Hypoxia culminates in methylation of DNA sequences in the promoter region of TM gene and increased the expression of DNMT1 and DNMT3B per se in primary HUVECs. Critical DNA methylation events were found to be compromised in high altitude DVT patients.

Vitamin D3-Induced Promotor Dissociation of PU.1 and YY1 Results in FcεRI Reduction on Dendritic Cells in Atopic Dermatitis.

Atopic dermatitis (AD) is a severe inflammatory skin disease. Langerhans cells and inflammatory dendritic epidermal cells (IDEC) are located in the epidermis of AD patients and contribute to the inflammatory processes. Both express robustly the high-affinity receptor for IgE, FcεRI, and thereby sense allergens. A beneficial role of vitamin D3 in AD is discussed to be important especially in patients with allergic sensitization. We hypothesized that vitamin D3 impacts FcεRI expression and addressed this in human ex vivo skin, in vitro Langerhans cells, and IDEC models generated from primary human precursor cells. We show in this article that biologically active vitamin D3 [1,25(OH)2-D3] significantly downregulated FcεRI at the protein and mRNA levels of the receptor's α-chain, analyzed by flow cytometry and quantitative RT-PCR. We also describe the expression of a functional vitamin D receptor in IDEC. 1,25(OH)2-D3-mediated FcεRI reduction was direct and resulted in impaired activation of IDEC upon FcεRI engagement as monitored by CD83 expression. FcεRI regulation by 1,25(OH)2-D3 was independent of maturation and expression levels of microRNA-155 and PU.1 (as upstream regulatory axis of FcεRI) and transcription factors Elf-1 and YY1. However, 1,25(OH)2-D3 induced dissociation of PU.1 and YY1 from the FCER1A promotor, evaluated by chromatin immunoprecipitation. We show that vitamin D3 directly reduces FcεRI expression on dendritic cells by inhibiting transcription factor binding to its promotor and subsequently impairs IgE-mediated signaling. Thus, vitamin D3 as an individualized therapeutic supplement for those AD patients with allergic sensitization interferes with IgE-mediated inflammatory processes in AD patients.

Abstract PR01: Structural mechanism of sequence-specific 5-methylcytosine (5mC) recognition by AP-1 transcription factors

Abstract DNA methylation is generally known to inhibit transcription factor binding. However, this notion has been challenged with identifications of several transcription factors that preferentially activate methylated promoters in specific sequences. AP-1 transcription factor (c-Jun/c-Fos heterodimer), a master regulator of a wide range of cancer-related genes, has been shown to bind a novel consensus sequence (5'-TGACTCG-3') in CpG methylation-dependent manner. In the fluorescence polarization-based DNA binding assay, both c-Jun/c-Fos heterodimer and c-Jun/c-Jun homodimer preferentially bind DNA containing the methylated consensus sequence compared to DNA with unmethylated sequence. Using a homodimer of c-Jun/c-Jun basic leucine zipper (bZIP) DNA binding domain, we have solved the X-ray structure of AP-1 in complex with an oligonucleotide containing the methylated consensus sequence. The structure reveals that a common motif conserved among bZIP proteins is involved in the specific recognition of methylated CpG by one monomer and TpG by the other monomer. Like 5mC, thymine (5-methyluracil) contains a methyl group at C5 position, and the similarity imposed by the methyl C5 preserves key structural features of DNA sequence recognized by AP-1. Our in vitro finding resonates with previous studies of AP-1-like transcription factors that preferentially bind methylated response elements. In light of such evidence, our characterization of AP-1 as a sequence-specific 5mCpG and TpG reader suggests a novel way in which DNA methylation may function to activate transcription in a methylation-dependent manner. Citation Format: Samuel Hong, Xiaodong Cheng. Structural mechanism of sequence-specific 5-methylcytosine (5mC) recognition by AP-1 transcription factors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR01.

Epigenetic upregulation of large-conductance Ca2+-activated K+ channel expression in uterine vascular adaptation to pregnancy.

Our previous study demonstrated that pregnancy increased large-conductance Ca(2+)-activated potassium channel β1 subunit (BKβ1) expression and large-conductance Ca(2+)-activated potassium channel activity in uterine arteries, which were abrogated by chronic hypoxia. The present study tested the hypothesis that promoter methylation/demethylation is a key mechanism in epigenetic reprogramming of BKβ1 expression patterns in uterine arteries. Ovine BKβ1 promoter of 2315 bp spanning from -2211 to +104 of the transcription start site was cloned, and an Sp1-380 binding site that contains CpG dinucleotide in its core binding sequences was identified. Site-directed deletion of the Sp1 site significantly decreased the BKβ1 promoter activity. Estrogen receptor-α bound to the Sp1 site through tethering to Sp1 and upregulated the expression of BKβ1. The Sp1 binding site at BKβ1 promoter was highly methylated in uterine arteries of nonpregnant sheep, and methylation inhibited transcription factor binding and BKβ1 promoter activity. Pregnancy caused a significant decrease in CpG methylation at the Sp1 binding site and increased Sp1 binding to the BKβ1 promoter and BKβ1 mRNA abundance. Chronic hypoxia during gestation abrogated this pregnancy-induced demethylation and upregulation of BKβ1 expression. The results provide evidence of a novel mechanism of promoter demethylation in pregnancy-induced reprogramming of large-conductance Ca(2+)-activated potassium channel expression and function in uterine arteries and suggest new insights of epigenetic mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and β-cell protection

Type 1 diabetes is due to destruction of pancreatic β-cells. Lysine deacetylase inhibitors (KDACi) protect β-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor--rather than global chromatin--hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100-120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing β-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1β + IFN-γ-induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.

Conformational modulation of DNA by polyamide binding: structural effects of f‐Im‐Py‐Im based derivatives on 5′‐ACGCGT‐3′

The DNA sequence 5'-ACGCGT-3' is in the core site of the Mlu 1 cell-cycle box, a transcriptional element in the promoter region of human Dbf4 gene that is highly correlated with a large number of aggressive solid cancers. The polyamide formamido-imidazole-pyrrole-imidazole-amine(+) (f-Im-Py-Im-Am(+) ) can target the minor groove of 5'-ACGCGT-3' as an antiparallel stacked dimer and has shown good activity in inhibiting transcription factor binding. Recently, f-Im-Py-Im-Am(+) derivatives that involve different orthogonally positioned substituents were synthesized to target the same binding site, and some of them have displayed improved binding and pharmacological properties. In this study, the gel electrophoresis-ligation ladders assay was used to evaluate the conformational effects of f-Im-Py-Im-Am(+) and derivatives on the target DNA, an essential factor for establishing the molecular basis of polyamide-DNA complexes and their transcription factor inhibition. The results show that the ACGCGT site in DNA has a relatively wide minor groove and a B-form like overall structure. After binding with f-Im-Py-Im-Am(+) derivatives, the DNA conformation is changed as indicated by the different mobilities in the gel. These conformational effects on DNA will at least help to point to the mechanism for the observed Mlu 1 inhibition activity of these polyamides. Therefore, modulating DNA transcription by locking the DNA shape or altering the minor groove geometry to affect the binding affinity of certain transcription factors is an attractive possible therapeutic mechanism for polyamides. Some of the substituents are charged with electrostatic interactions with DNA phosphate groups, and their charge effects on DNA gel mobility have been observed.

Chronic Hypoxia During Gestation Causes Epigenetic Repression of the Estrogen Receptor-α Gene in Ovine Uterine Arteries via Heightened Promoter Methylation

Estrogen receptor-α (ERα) plays a key role in the adaptation of increased uterine blood flow in pregnancy. Chronic hypoxia is a common stress to maternal cardiovascular homeostasis and causes increased risk of preeclampsia. Studies in pregnant sheep demonstrated that hypoxia during gestation downregulated ERα gene expression in uterine arteries. The present study tested the hypothesis that hypoxia causes epigenetic repression of the ERα gene in uterine arteries via heightened promoter methylation. Ovine ERα promoter of 2035 bp spanning from −2000 to +35 of the transcription start site was cloned. No estrogen or hypoxia-inducible factor response elements were found at the promoter. Two transcription factor binding sites, USF −15 and Sp1 −520 , containing CpG dinucleotides were identified, which had significant effects on the promoter activity. The USF element binds transcription factors USF1 and USF2, and the Sp1 element binds Sp1, as well as ERα through Sp1. Deletion of the Sp1 site abrogated 17β-estradiol–induced increase in the promoter activity. In normoxic control sheep, CpG methylation at the Sp1 but not the USF site was significantly decreased in uterine arteries of pregnant as compared with nonpregnant animals. In pregnant sheep exposed to long-term high-altitude hypoxia, CpG methylation at both Sp1 and USF sites in uterine arteries was significantly increased. Methylation inhibited transcription factor binding and the promoter activity. The results provide evidence of hypoxia causing heightened promoter methylation and resultant ERα gene repression in uterine arteries and suggest new insights of molecular mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

DNA cytosine methylation in the Bovine Leukemia Virus promoter is associated with latency in a Lymphoma-derived B-cell line : potential involvement of direct inhibition of CREB/CREM/ATF binding

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Occlusion of Regulatory Sequences by Promoter Nucleosomes In Vivo

Nucleosomes are believed to inhibit DNA binding by transcription factors. Theoretical attempts to understand the significance of nucleosomes in gene expression and regulation are based upon this assumption. However, nucleosomal inhibition of transcription factor binding to DNA is not complete. Rather, access to nucleosomal DNA depends on a number of factors, including the stereochemistry of transcription factor-DNA interaction, the in vivo kinetics of thermal fluctuations in nucleosome structure, and the intracellular concentration of the transcription factor. In vitro binding studies must therefore be complemented with in vivo measurements. The inducible PHO5 promoter of yeast has played a prominent role in this discussion. It bears two binding sites for the transcriptional activator Pho4, which at the repressed promoter are positioned within a nucleosome and in the linker region between two nucleosomes, respectively. Earlier studies suggested that the nucleosomal binding site is inaccessible to Pho4 binding in the absence of chromatin remodeling. However, this notion has been challenged by several recent reports. We therefore have reanalyzed transcription factor binding to the PHO5 promoter in vivo, using ‘chromatin endogenous cleavage’ (ChEC). Our results unambiguously demonstrate that nucleosomes effectively interfere with the binding of Pho4 and other critical transcription factors to regulatory sequences of the PHO5 promoter. Our data furthermore suggest that Pho4 recruits the TATA box binding protein to the PHO5 promoter.

The dietary flavonoid apigenin blocks phorbol 12-myristate 13-acetate-induced COX-2 transcriptional activity in breast cell lines

Cyclooxygenase (COX)-2 is the inducible isozyme catalyzing the conversion of arachidonic acid to prostanoids. Its expression has been linked to the process of carcinogenesis, including tumorigenesis of the breast. Apigenin (APG) is a flavone commonly found in fruit and vegetables, and is shown to be a potential modulator in inflammatory diseases. This study examined the potential suppressive effect of the flavone on phorbol ester-induced COX-2 expression in the breast cell lines MCF-10A and MCF-7. Real-time PCR and/or Western blotting indicated that APG in micromolar range significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in these breast cells. APG treatment reduced the amount of phospho-mitogen-activated protein kinase (MAPK) ERK-1/2, but it did not alter the activity of PKC. Activated ERKs might trigger the transactivation of AP-1 or CRE, which can be located at COX-2 promoter region (−72/−53). Reporter gene assay as well as electrophoretic mobility shift assays (EMSA) illustrated that APG inhibited transcription factor binding at this region in a dose-dependent manner. This study showed that APG down-regulated PMA-induced COX-2 expression in breast cells without affecting PKC activity. These findings could provide a scientific basis for developing APG nutraceutical against breast carcinogenesis.

DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line: POTENTIAL INVOLVEMENT OF DIRECT INHIBITION OF cAMP-RESPONSIVE ELEMENT (CRE)-BINDING PROTEIN/CRE MODULATOR/ACTIVATION TRANSCRIPTION FACTOR BINDING

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

DNA methylation determination by liquid chromatography-tandem mass spectrometry using novel biosynthetic [U-15N]deoxycytidine and [U-15N]methyldeoxycytidine internal standards

Methylation of the promoter CpG regions regulates gene transcription by inhibiting transcription factor binding. Deoxycytidine methylation may regulate cell differentiation, while aberrations in the process may be involved in cancer etiology and the development of birth defects (e.g. neural tube defects). Similarly, nutritional deficiency and certain nutragenomic interactions are associated with DNA hypomethylation. While LC-MS has been used previously to measure percentage genomic deoxycytidine methylation, a lack of a secure source of internal standards and the need for laborious and time-consuming DNA digestion protocols constitute distinct limitations. Here we report a simple and inexpensive protocol for the biosynthesis of internal standards from readily available precursors. Using these biosynthetic stable-isotopic [U-15N]-labeled internal standards, coupled with an improved DNA digestion protocol developed in our lab, we have developed a low-cost, high-throughput (>500 samples in 4 days) assay for measuring deoxycytidine methylation in genomic DNA. Inter- and intraassay variation for the assay (%RSD, n = 6) was <2.5%.

High Selenium Reduces NF-κB-Regulated Gene Expression in Uninduced Human Prostate Cancer Cells

Nuclear factor kappa B (NF-κB) induces expression of antiapoptotic and pro-inflammatory genes and is constitutively activated in prostate cancer. We tested the hypothesis that a biologically and physiologically relevant form and concentration of selenium (Se) may alter NF-κ B activation in early prostate cancer cells in the absence of exogenously added inducers of the NF-κB pathway. LNCaP cells were cultured in medium without added tumor necrosis factor alpha or lipopolysaccharide but with methylseleninic acid added to provide final concentrations of Se of 30 nM–7.6 μM. Compared to 50 nM Se, treatment with 7.6 μM Se virtually eliminated NF-κB binding to its DNA response element and reduced transcription rates and mRNA levels by half for NF-κB-regulated genes. There were no differences due to Se in tyrosine phosphorylation, inhibitor of kappa B alpha (IκBα) levels, or NF-κB translocation from cytosol to nucleus. The observation in these basal, unstimulated cells of altered NF-κB binding to DNA in the absence of effects on the NF-κB activation pathway suggests an interaction of Se with the NF-κB protein or an effect on recruitment of NF-κB coactivators or corepressors. Inhibition of transcription factor binding and anti-apoptotic gene expression may be one mechanism for the chemopreventive effects of Se against prostate cancer.

Suppression of thymidine phosphorylase expression by promoter methylation

21100 Background: Thymidine phosphorylase (TP), an important enzyme in the pyrimidine salvage pathway, is also responsible for the activation of capecitabine (CAPE), a 5-FU prodrug. TP expression has been associated with response to CAPE. The regulatory mechanisms of TP expression in human tumor cells remain unclear. Since promoter methylation of CpG sites is a known mechanism of reversible silencing of gene expression, we investigated whether TP promoter methylation is involved in the regulation of TP expression. Methods: TP mRNA levels were determined in 5 cancer cells (SKBR-3, 786-O, HT-29, MDA-231, DLD-1) by Real- Time Quantitative PCR (RT-Q-PCR).The degree of methylation in these cells was identified by examining methylated CpG sites with restriction PCR and bisulfite sequencing. To analyze the TP promoter region, two fragments were PCR amplified from genomic DNA. Minimal promoter activity was determined by Luciferase reporter assays, and DNA-protein interactions were evaluated by Electrophoretic Mobility Shift Assays (EMSA). Results: SKBR-3 cells showed high TP expression, while DLD-1 cells did not express TP, as confirmed by examination of TP mRNA, protein, and enzyme activity. 786-O, HT29, and MDA-231 cells demonstrated intermediate TP expression. Methylation in the promoter, intron 1 and exon1 regions of the TP gene in SKBR-3 was lacking, while in DLD-1 cells, extensive promoter methylation was observed. Treatment of DLD-1 and SKBR-3 cells with the methylation-inhibitor, 5-aza-2-dC, resulted in a concentration-dependent increase in TP mRNA as well as protein levels in DLD-1 but not SKBR-3 cells. Trichostatin-A (TSA), a histone deacetylase inhibitor, improved the 5-aza-2dC induced TP reactivation. Gel shift assays demonstrated that methylation of the TP promoter significantly inhibited transcription factor binding. Supershift Analyses suggest that the Sp1 transcription factor has a role in the regulation of TP expression and to a lesser degree, so does Sp3. Conclusions: These results establish methylation of site-specific CpG in the TP promoter to be a putative mechanism of down-regulated TP expression in cancer cells. Insight into the TP regulatory mechanisms may have implications in CAPE therapy optimization and in modulating prognosis of cancer patients treated with CAPE. No significant financial relationships to disclose.

Inhibition of IFN-γ transcription by site-specific methylation during T helper cell development

Polarization of naïve CD4 T cells into T helper type 2 (TH2) cells is characterized by expression of IL-4 and silencing of IFN-gamma. Here we show that during TH2 polarization, the DNA methyltransferase Dnmt3a is recruited to the IFN-gamma promoter and correspondingly the promoter undergoes progressive de novo methylation. Notably, the CpG located at the -53 position becomes methylated rapidly and this methylation inhibits ATF2/c-Jun and CREB transcription factor binding in vitro. In vivo, the same factors bind to the unmethylated IFN-gamma promoter in T helper type 1 (TH1) cells but not the methylated IFN-gamma promoter in TH2 cells. Furthermore, methylation at the -53 CpG alone is sufficient to inhibit the IFN-gamma promoter-driven reporter gene expression in a TH1 cell line. These findings suggest that rapid methylation of the evolutionarily conserved -53 CpG by Dnmt3a may suppress IFN-gamma transcription in developing TH2 cells by directly inhibiting transcription factor binding.

Differential Regulation of the Alcohol Dehydrogenase 1B (ADH1B) and ADH1C Genes by DNA Methylation and Histone Deacetylation

The human class I alcohol dehydrogenase (ADH) genes (ADH1A, ADH1B, and ADH1C) differ in expression during development and in various tissues. They are repressed in the HepG2 human hepatoma cell line. We hypothesized that epigenetic modifications play a role in this repression and that class I ADH gene expression would be enhanced upon global inhibition of DNA methylation and histone deacetylation. Southern blotting was used to assess the methylation status of each class I ADH gene. HepG2 and HeLa cells were treated with either the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the histone deacetylase inhibitor Trichostatin A (TSA), or both in combination, and class I ADH gene expression was analyzed. Chromatin immunoprecipitation assays were performed to analyze histone H3 acetylation. Transient transfections and gel mobility shift assays were used to analyze the role that methylation plays in inhibiting transcription factor binding and promoter function. We show that the upstream regions of ADH1A, ADH1B, and ADH1C are methylated in HepG2 cells. 5-Aza-2'-deoxycytidine treatment enhanced expression of both ADH1B and ADH1C. Trichostatin A treatment elevated expression of ADH1C. ADH1A expression was not stimulated by either 5-aza-dC or TSA. H3 histones associated with a methylated upstream region of ADH1B were hyperacetylated in TSA-treated, but not in 5-aza-dC-treated, HepG2 cells. A methylated upstream region of ADH1C achieved histone H3 hyperacetylation upon either 5-aza-dC or TSA treatment. Methylation of the ADH1B proximal promoter in vitro decreased its activity to 54% and inhibited the binding of the upstream stimulatory factor. These findings suggest that the class I ADH genes are regulated by epigenetic mechanisms in human hepatoma cells. The temporal and tissue-specific expression of these genes may in part result from differences in epigenetic modifications and the availability of key transcription factors.

BMP4 Regulates Pancreatic Progenitor Cell Expansion through Id2

Inhibitor of DNA binding (Id) proteins bind to and inhibit the function of basic helix-loop-helix (bHLH) transcription factors including those that regulate pancreatic development. Moreover, bone morphogenetic proteins (BMPs) regulate the expression of Ids. We hypothesized that BMP4 and Id proteins play a role in the expansion and differentiation of epithelial progenitor cells. We demonstrate that BMP4 induces the expression of Id2 along with the expansion of AR42J pancreatic epithelial cells. Furthermore, neutralization of BMP4 significantly reduced duct epithelial cell expansion in a mouse model of islet regeneration. BMP4 stimulation promotes Id2 binding to the bHLH transcription factor NeuroD, which is required for the differentiation of pancreatic islet cells. Therefore, our results indicate that BMP4 stimulation blocks the differentiation of endocrine progenitor cells and instead promotes their expansion thereby revealing a novel paradigm of signaling explaining the balance between expansion and differentiation of pancreatic duct epithelial progenitors. Understanding the mechanisms of BMP and Id function elucidates a key step during pancreas embryogenesis, which is important knowledge for expanding pancreatic progenitors in vitro.

Functional Characterization of the BCL11B Breakpoint Region Reveals Multiple Enhancers at Dnase-I Hypersensitive Sites Some of Which Control NK-Family Homeobox Genes in T-ALL Cells with t(5;14)(q35;q32).

The zinc-finger transcription factor BCL11B (14q32) is a key translocation target in pediatric T-cell acute lymphoblastic leukemia (T-ALL) where it ectopically activates NK-family homeobox genes, chiefly TLX3, or NKX2-5, via the recurrent t(5;14)(q35;q32). However, the mechanism underlying leukemic activation by BCL11B remains obscure. Breakpoints at 14q32 are dispersed over about 1.2 Mbp downstream of BCL11B amid a non-coding genomic “desert”. Detailed cytogenetic analysis of T-ALL cell lines, including one carrying a complex double-insertion enabled the putative breakpoint target zone to be narrowed to ~300 kbp covering the central and distal parts of the 3′-BCL11B region. Interestingly, Crawford et al. (PNAS 101: 992–7, 2004) have identified a series of DNase-I hypersensitive sites (DHS) in T-cells amid this genomic desert including a unique cluster near the most distal breakpoint to serve as candidate BCL11B transcriptional enhancers. Accordingly, we used the TRANSFAC database to identify multiple transcription factor (TF) binding site sequences nearby these DHS. To investigate the role of DHS in activating homeobox transcription in T-ALL, we designed double-stranded 26-mer oligos matching each of 9 DHS which were used to transfect a T-ALL cell line (PEER) in which NKX2-5 is inserted about 1 Mbp downstream of BCL11B and thereby activated. Such oligos may inhibit TF binding by directly blocking access to DHS or act as decoys to sequester TF. We found that oligos targeting DHS in the central and distal regions of 3′-BCL11B, close to the insertion junctions in PEER cells, were effective in down-regulating NKX2-5 transcription in a dose dependent manner. In contrast, oligos targeting DHS near 3′-BCL11B were able to down-regulate BCL11B but not NKX2-5. Treatments with neither mutated DHS oligos, nor other control oligos caused inhibition. Transcription of ESTs present at 3′-BCL11B was unaffected by DHS-oligo transfection indicating specific targeting of BCL11B or NKX2-5 by their respective DHS. Analysis of t(5;14) T-ALL cell lines by Halo-FISH, showed the selective homing of 3′-BCL11B chromatin to the nuclear matrix when juxtaposed with NKX2-5, whereas 3′-BCL11B chromatin present on non-participant chromosomes was extruded to surrounding halos inimical to transcription. Taken together, our data show the presence of multiple DHS covering the ~1.2 Mbp breakpoint dispersal region at 3′-BCL11B in T-ALL cells which appear to serve as enhancers of BCL11B or nearby translocated NK-family homeobox genes. These multiple enhancers may be required for the precise timing of BCL11B transcription which is critical to thymocyte development - a question which will be addressed by planned future studies. As well as revealing novel aspects underlying the transcriptional control of BCL11B in T-cells, our data highlight a potential therapeutic target in leukemia within “junk DNA”.