Transcription Factor Recognition Sequences Research Articles

Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus

Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci.

DNA Methylation of the Angiotensinogen Gene, AGT, and the Aldosterone Synthase Gene, CYP11B2 in Cardiovascular Diseases.

Angiotensinogen (AGT) and aldosterone play key roles in the regulation of blood pressure and are implicated in the pathogenesis of cardiovascular diseases. DNA methylation typically acts to repress gene transcription. The aldosterone synthase gene CYP11B2 is regulated by angiotensin II and potassium. DNA methylation negatively regulates AGT and CYP11B2 expression and dynamically changes in response to continuous promoter stimulation of each gene. High salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT-enhancer-binding-protein (CEBP) sites of the ATG promoter region, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue and heart. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in salt-sensitive hypertensive rats. Salt-dependent hypertension may be partially affected by increased cardiac AGT expression. CpG dinucleotides in the CYP11B2 promoter are hypomethylated in aldosterone-producing adenomas. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminish their DNA-binding activity. The methylated CpG-binding protein MECP2 interacts directly with the methylated CYP11B2 promoter. Low salt intake and angiotensin II infusion lead to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with the angiotensin II type 1 receptor antagonist decreases CYP11B2 expression and leads to DNA hypermethylation. A close association between low DNA methylation and increased CYP11B2 expression are seen in the hearts of patients with hypertrophic cardiomyopathy. These results indicate that epigenetic regulation of both AGT and CYP11B2 contribute to the pathogenesis of cardiovascular diseases.

The Transcription Factor ERG Regulates Super-Enhancers Associated With an Endothelial-Specific Gene Expression Program.

The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG's homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms from genome-wide association studies lie within noncoding regions and perturb transcription factor recognition sequences in relevant cell types. Analysis of genome-wide association studies data shows significant enrichment of risk variants for cardiovascular disease and other diseases, at ERG endothelial enhancers and super-enhancers. The transcription factor ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of cardiovascular disease-associated single nucleotide polymorphisms at ERG super-enhancers suggests that ERG-dependent transcription modulates disease risk.

Epigenetic Regulation of Aldosterone Synthase Gene by Sodium and Angiotensin II.

Background DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We demonstrate that stimulatory signals can change the DNA methylation status around transcription factor binding sites and a transcription start site and activate expression of the aldosterone synthase gene (CYP11B2).Methods and Results DNA methylation of CYP11B2 was analyzed in aldosterone‐producing adenomas, nonfunctioning adrenal adenomas, and adrenal glands and compared with the gene expression levels. CpG dinucleotides in the CYP11B2 promoter were found to be hypormethylated in tissues with high expression, but not in those with low expression, of CYP11B2. Methylation of the CYP11B2 promoter fused to a reporter gene decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminished their DNA‐binding activity. A methylated‐CpG‐binding protein MECP2 interacted directly with the methylated CYP11B2 promoter. In rats, low salt intake led to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with angiotensin II type 1 receptor antagonist decreased CYP11B2 expression and led to DNA hypermethylation.Conclusions DNA demethylation may switch the phenotype of CYP11B2 expression from an inactive to an active state and regulate aldosterone biosynthesis.

Abstract 5242: Further evidence that the DNA-interactive Pyrrolobenzodiazepine (PBD) Dimer SJG-136 works through a transcription factor inhibition mechanism

Abstract The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents. The PBD dimer SJG-136 has currently been investigated in Phase II clinical trials in ovarian cancer and leukaemia in the UK and the USA. More recently, PBD dimer analogues are being attached to tumour-targeting antibodies to create Antibody-Drug Conjugates (ADCs), some which are now in Phase 3 clinical trials with many others in pre-clinical and clinical development. Transcription factors (TFs) are sequence-specific DNA-interacting proteins that bind to consensus DNA sequences, thereby controlling transcription. TFs regulate processes such as cell differentiation, proliferation and apoptosis. The interaction of a small-molecule with the consensus DNA recognition sequences of TFs can prevent a TF from interacting with its cognate sequence, thereby inhibiting the expression of genes critical for the survival and proliferation of cancer cells. There is growing evidence that PBDs may exert, at least in part, their pharmacological effect through TF inhibition in addition to the arrest of the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. For this reason, there is now interest in using PBDs as the basis for a small-molecule strategy to target specific DNA sequences for TF inhibition as a novel anticancer therapy. We have developed a reversed-phase HPLC/MS method as a tool to evaluate the interaction of DNA-binding PBD molecules with oligonucleotides of varying lengths and sequences. Using this methodology, we have demonstrated that the PBD dimer SJG-136 binds to the cognate sequences of the oncogenic transcription factors NF-κB, EGR-1, AP-1 and STAT3. Surprisingly, significant differences in the rate and extent of adduct formation between the different cognate sequences were observed which may explain, at least in part, the differences in potency of SJG-136 in various tumour cell lines. Furthermore, an RT PCR study has been carried out using the human tumour cell lines MDA-MB-231 (breast) and HT-29 (colon) to see whether these transcription factors are affected in vitro. The results were consistent with the HPLC-MS studies in that SJG-136 was shown to significantly down-regulate a number of AP-1- and STAT3-dependent genes such as Bcl-2, VEGF, p53 and survivin. These findings add significantly to knowledge of the mechanism of action of SJG-136 and related PBD compounds, and could be relevant for the correct interpretation of clinical activity of molecules of this type both as standalone agents and as ADC payloads. Citation Format: Julia Mantaj, Paul J. Jackson, David E. Thurston, Khondaker Miraz Rahman. Further evidence that the DNA-interactive Pyrrolobenzodiazepine (PBD) Dimer SJG-136 works through a transcription factor inhibition mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5242. doi:10.1158/1538-7445.AM2017-5242

A Novel System for the Quantification of the ADCC Activity of Therapeutic Antibodies

Novel ADCC effector cells expressing the V-variant or F-variant of FcγRIIIa (CD16a) and firefly luciferase under the control of a chimeric promoter incorporating recognition sequences for the principal transcription factors involved in FcγRIIIa signal transduction, together with novel target cells overexpressing a constant high level of the specific antigen recognized by rituximab, trastuzumab, cetuximab, infliximab, adalimumab, or etanercept, confer improved sensitivity, specificity, and dynamic range in an ADCC assay relative to effector cells expressing a NFAT-regulated reporter gene and wild-type target cells. The effector cells also contain a normalization gene rendering ADCC assays independent of cell number or serum matrix effects. The novel effector and target cells in a frozen thaw-and-use format exhibit low vial-to-vial and lot-to-lot variation in their performance characteristics reflected by CVs of 10% or less. Homologous control target cells in which the specific target gene has been invalidated by genome editing providing an ideal control and a means of correcting for nonspecific effects were observed with certain samples of human serum. The novel effector cells and target cells expressing noncleavable membrane-bound TNFα have been used to quantify ADCC activity in serum from patients with Crohn's disease treated with infliximab and to relate ADCC activity to drug levels.

Expression of metastasis suppressor gene AES driven by a Yin Yang (YY) element in a CpG island promoter and transcription factor YY2.

We recently found that the product of the AES gene functions as a metastasis suppressor of colorectal cancer (CRC) in both humans and mice. Expression of amino‐terminal enhancer of split (AES) protein is significantly decreased in liver metastatic lesions compared with primary colon tumors. To investigate its downregulation mechanism in metastases, we searched for transcriptional regulators of AES in human CRC and found that its expression is reduced mainly by transcriptional dysregulation and, in some cases, by additional haploidization of its coding gene. The AES promoter‐enhancer is in a typical CpG island, and contains a Yin‐Yang transcription factor recognition sequence (YY element). In human epithelial cells of normal colon and primary tumors, transcription factor YY2, a member of the YY family, binds directly to the YY element, and stimulates expression of AES. In a transplantation mouse model of liver metastases, however, expression of Yy2 (and therefore of Aes) is downregulated. In human CRC metastases to the liver, the levels of AES protein are correlated with those of YY2. In addition, we noticed copy‐number reduction for the AES coding gene in chromosome 19p13.3 in 12% (5/42) of human CRC cell lines. We excluded other mechanisms such as point or indel mutations in the coding or regulatory regions of the AES gene, CpG methylation in the AES promoter enhancer, expression of microRNAs, and chromatin histone modifications. These results indicate that Aes may belong to a novel family of metastasis suppressors with a CpG‐island promoter enhancer, and it is regulated transcriptionally.

A Role for the Transcription Factor Nk2 Homeobox 1 in Schizophrenia: Convergent Evidence from Animal and Human Studies.

Schizophrenia is a highly heritable disorder with diverse mental and somatic symptoms. The molecular mechanisms leading from genes to disease pathology in schizophrenia remain largely unknown. Genome-wide association studies (GWASs) have shown that common single-nucleotide polymorphisms associated with specific diseases are enriched in the recognition sequences of transcription factors that regulate physiological processes relevant to the disease. We have used a “bottom-up” approach and tracked a developmental trajectory from embryology to physiological processes and behavior and recognized that the transcription factor NK2 homeobox 1 (NKX2-1) possesses properties of particular interest for schizophrenia. NKX2-1 is selectively expressed from prenatal development to adulthood in the brain, thyroid gland, parathyroid gland, lungs, skin, and enteric ganglia, and has key functions at the interface of the brain, the endocrine-, and the immune system. In the developing brain, NKX2-1-expressing progenitor cells differentiate into distinct subclasses of forebrain GABAergic and cholinergic neurons, astrocytes, and oligodendrocytes. The transcription factor is highly expressed in mature limbic circuits related to context-dependent goal-directed patterns of behavior, social interaction and reproduction, fear responses, responses to light, and other homeostatic processes. It is essential for development and mature function of the thyroid gland and the respiratory system, and is involved in calcium metabolism and immune responses. NKX2-1 interacts with a number of genes identified as susceptibility genes for schizophrenia. We suggest that NKX2-1 may lie at the core of several dose dependent pathways that are dysregulated in schizophrenia. We correlate the symptoms seen in schizophrenia with the temporal and spatial activities of NKX2-1 in order to highlight promising future research areas.

Exonic Transcription Factor Binding Directs Codon Choice and Affects Protein Evolution

Genomes contain both a genetic code specifying amino acids and a regulatory code specifying transcription factor (TF) recognition sequences. We used genomic deoxyribonuclease I footprinting to map nucleotide resolution TF occupancy across the human exome in 81 diverse cell types. We found that ~15% of human codons are dual-use codons ("duons") that simultaneously specify both amino acids and TF recognition sites. Duons are highly conserved and have shaped protein evolution, and TF-imposed constraint appears to be a major driver of codon usage bias. Conversely, the regulatory code has been selectively depleted of TFs that recognize stop codons. More than 17% of single-nucleotide variants within duons directly alter TF binding. Pervasive dual encoding of amino acid and regulatory information appears to be a fundamental feature of genome evolution.

DNA methylation alone does not cause most cell-type selective transcription factor binding

Background DNA methylation at vertebrate promoters is associated with the repression of gene expression and is required for mammalian development [1]. The master genome regulator and transcription factor CTCF canonically exhibits methylation-sensitive binding in vitro ,a nd its in vivo occupancy across cell types anticorrelates with methylation at 41 % of sites genome-wide [2]. However, the genome harbors hundreds of thousands of CTCF recognition sequences, the majority of which are unbound in any cell type. Although most unbound recognition sequences harbor methylation in a given cell type, the causal role of DNA methylation in the abrogation of transcription factor binding at these potential binding sites is largely unknown [3,4]. Materials and methods Here we perform genome-wide occupancy profiling after both stable genetic and transient chemical inhibition of DNA methyltransferases in HCT116 cells lacking functional DNMT1 and DNMT3b and K562 erythroleukemia cells treated with 5-aza-2-deoxycytidine. Results We show that the vast majority of susceptible binding sites remain unoccupied upon depletion of DNA methylation. Stable loss of methylation in HCT116 cells does result in a minor increase of several thousand binding sites, largely corresponding to selective reactivation of binding sites from other cell types, especially from other malignant cell lines. Chemical inhibition in K562 cells results in a smaller but reproducible set of several hundred reactivated sites, the majority of which are corroborated by the genetic data. The narrow extent of reactivation implies that the majority of transcription factor recognition sequences are not competent for binding even in the absence of methylation, and that methylation accounts for only a modest fraction of cell-selective binding. These results offer new insight into the establishment of the global regulatory landscape, and offer an important perspective on the interpretation of disease- and trait-associated methylation differences in humans.

AtMYB2 transcription factor can interact with the CMO promoter and regulate its downstream gene expression

The pC5 promoter, a region of the choline monooxygenase (CMO) promoter, contains an AtMYB2 transcription factor recognition sequence, TAACCA, and we have examined the interaction between AtMYB2 and the pC5 promoter. The AtMYB2 gene was cloned from Arabidopsis, expressed in Escherichia coli and transferred into pC5-GUS transgenic tobacco plants. Using an electrophoretic mobility shift assay, the AtMYB2 fusion protein binds to the TAACCA sequence in the pC5 promoter. As GUS activity was higher in pC5-GUS/AtMYB2 transgenic tobacco than in pC5-GUS plants, the AtMYB2 protein can bind to the CMO promoter in vitro and activate the transcription of the GUS reporter gene in vivo. The AtMYB2 transcription factor can therefore interact with the CMO promoter directly and regulate its downstream gene expression.

Widespread Site-Dependent Buffering of Human Regulatory Polymorphism

The average individual is expected to harbor thousands of variants within non-coding genomic regions involved in gene regulation. However, it is currently not possible to interpret reliably the functional consequences of genetic variation within any given transcription factor recognition sequence. To address this, we comprehensively analyzed heritable genome-wide binding patterns of a major sequence-specific regulator (CTCF) in relation to genetic variability in binding site sequences across a multi-generational pedigree. We localized and quantified CTCF occupancy by ChIP-seq in 12 related and unrelated individuals spanning three generations, followed by comprehensive targeted resequencing of the entire CTCF–binding landscape across all individuals. We identified hundreds of variants with reproducible quantitative effects on CTCF occupancy (both positive and negative). While these effects paralleled protein–DNA recognition energetics when averaged, they were extensively buffered by striking local context dependencies. In the significant majority of cases buffering was complete, resulting in silent variants spanning every position within the DNA recognition interface irrespective of level of binding energy or evolutionary constraint. The prevalence of complex partial or complete buffering effects severely constrained the ability to predict reliably the impact of variation within any given binding site instance. Surprisingly, 40% of variants that increased CTCF occupancy occurred at positions of human–chimp divergence, challenging the expectation that the vast majority of functional regulatory variants should be deleterious. Our results suggest that, even in the presence of “perfect” genetic information afforded by resequencing and parallel studies in multiple related individuals, genomic site-specific prediction of the consequences of individual variation in regulatory DNA will require systematic coupling with empirical functional genomic measurements.

Transcription Factor-recognition Sequences Potentially Involved in Modulation of Gene Expression after Exposure to Low-dose-rate γ-rays in the Mouse Liver

In vivo modulation of gene expression profiles after low-dose and low-dose-rate irradiation has been observed in a variety of experimental systems. However, few studies actually investigated the underlying mechanisms for these genetic responses. In this study, we used pre-existing microarray data and searched for gene modulations in response to long-term, low-dose-rate irradiation. Nucleotide sequences in the neighboring region of the up-regulated, down-regulated, and unaffected genes were retrieved from the Entrez Gene database, and recognition sequences for transcription factors (TFs) were searched using the TFSEARCH database. As a result, we suggested 21 potential TF-binding sites with significantly different incidence between the three gene groups (up-regulated, down-regulated and unaffected gene groups). The binding sites for sterol regulatory element-binding protein 1 (SREBP-1), aryl hydrocarbon receptor (AhR/Ar) and olfactory 1 (Olf-1) were suggested to be involved in up-regulation, while the binding sites for glucocorticoid receptor (GR(GGTACAANNT GTYCTK) ) and hepatocyte nuclear factor 1 (HNF-1) were suggested to be involved in down-regulation of the genes. In addition, the binding sites for activating enhancer-binding protein 4 (AP-4), nuclear factor-κB (NFκB), GR (NNNNNNCNNTNTGTNCTNN) and early growth response 3 (Egr-3) were correlated with modulation of gene expression regardless of the direction of modulation. Our results suggest that these TF-binding sites are involved in gene modulations after long-term continuous irradiation with low-dose-rate γ rays. GR and/or SREBP-1 might be associated with the altered metabolic process observed in liver after exposure to low-dose-rate irradiation.

Early demethylation of non-CpG, CpC-rich, elements in the myogenin 5’-flanking region

The dynamic changes and structural patterns of DNA methylation of genes without CpG islands are poorly characterized. The relevance of CpG to the non-CpG methylation equilibrium in transcriptional repression is unknown. In this work, we analyzed the DNA methylation pattern of the 5’-flanking of the myogenin gene, a positive regulator of muscle differentiation with no CpG island and low CpG density, in both C2C12 muscle satellite cells and embryonic muscle. Embryonic brain was studied as a non-expressing tissue. High levels of both CpG and non-CpG methylation were observed in non-expressing experimental conditions. Both CpG and non-CpG methylation rapidly dropped during muscle differentiation and myogenin transcriptional activation, with an active demethylation dynamics. Non-CpG demethylation occurred more rapidly than CpG demethylation. Demethylation spread from initially highly methylated short CpC-rich elements to a virtually unmethylated status. These short elements have a high CpC content and density, share some motifs and largely coincide with putative recognition sequences of some differentiation-related transcription factors. Our findings point to a dynamically controlled equilibrium between CpG and non-CpG active demethylation in the transcriptional control of tissue-specific genes. The short CpC-rich elements are new structural features of the methylation machinery, whose functions may include priming the complete demethylation of a transcriptionally crucial DNA region.

Cloning and functional characterization of the rat α2B-adrenergic receptor gene promoter region: Evidence for binding sites for erythropoiesis-related transcription factors GATA1 and NF-E2

In the rat, the α2B-adrenergic receptor (α2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of α2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of α2B-AR expression, a genomic fragment containing 2.8 kb of the 5′-flanking region, the ORF and approximately 20 kb of the 3′-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5′-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332 bp fragment (−732/−401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0 kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for α2B-AR expression during fetal life.

MOLECULAR MECHANISMS REGULATING HUMANCYP4B1LUNG-SELECTIVE EXPRESSION

Lung-selective cytochrome P450 expression is well recognized; however, little is known regarding regulatory mechanisms. To address this knowledge gap, transient expression of CYP4B1/luciferase constructs was used to identify a proximal, positively acting regulatory element, position -139 to -45, that functioned in all cells examined; a negatively acting element, position -457 to -216, that only functioned in HepG2 hepatoblastoma cells; and a distal, positively acting element, position -1087 to -1008, that functioned in A549 or BEAS-2B lung-derived cells but not HepG2 cells or 293 kidney-derived cells. Competitive electrophoretic mobility shift assays further localized specific A549, but not HepG2, nuclear protein binding to two sites within the distal element, CYP4B1 position -1052 to -1042 and -1026 to -1008. Several potential lung-selective transcription factor recognition sequences were identified within these elements. However, attempts to identify specific factor(s) were unsuccessful. In contrast, in vitro DNA/protein binding assays combined with transient expression and mutagenesis studies identified two functional Sephadex protein/Krüppel-like factor families of transcription factor sites within the proximal element (position -118 to -114 and position -77 to -73) that bound both Sephadex protein 1 (Sp1) and Sephadex protein 3 (Sp3) in vitro. Furthermore, Sp1-dependent synergistic regulation was observed in A549 cells involving the proximal and distal regulatory elements. Chromatin immunoprecipitation assays demonstrated binding of neither Sp1 nor Sp3 to the CYP4B1 proximal element in human liver tissue, whereas selective Sp1 binding was observed in human lung tissue. Thus, the composite findings are consistent with both the proximal Sp1 elements and the distal regulatory element acting to synergistically control CYP4B1 lung-selective expression.

Cdx Binding Determines the Timing of Enhancer Activation in Postnatal Duodenum

In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.

Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivo upon rhinovirus infection

Human rhinovirus (HRV) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease (COPD) and are associated with lymphocytic infiltration of the airways. We demonstrate that infection of primary cultures of human airway epithelial cells, or of the BEAS-2B human bronchial epithelial cell line, with human rhinovirus type 16 (HRV-16) induces expression of CXCL10 [IFN-gamma-inducible protein 10 (IP-10)], a ligand for the CXCR3 receptor found on activated type 1 T lymphocytes and natural killer cells. IP-10 mRNA reached maximal levels 24 h after HRV-16 infection then declined, whereas protein levels peaked 48 h after infection with no subsequent new synthesis. Cytosolic levels of AU-rich factor 1, a protein associated with mRNA destabilization, increased beginning 24 h after HRV-16 infection. Generation of IP-10 required virus capable of replication but was not dependent on prior induction of type 1 interferons. Transfection of synthetic double-stranded RNA into epithelial cells induced robust production of IP-10, whereas transfection of single-stranded RNA had no effect. Induction of IP-10 gene expression by HRV-16 depended upon activation of NF-kappaB, as well as other transcription factor recognition sequences further upstream in the IP-10 promoter. In vivo infection of human volunteers with HRV-16 strikingly increased IP-10 protein in nasal lavages during symptomatic colds. Levels of IP-10 correlated with symptom severity, viral titer, and numbers of lymphocytes in airway secretions. Thus IP-10 may play a role in the pathogenesis of HRV-induced colds and in HRV-induced exacerbations of COPD and asthma.

Epigenetic regulation of 11 beta-hydroxysteroid dehydrogenase type 2 expression.

The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11 beta-hydroxyglucocorticoids. Variable activity of 11 beta HSD2 is relevant for blood pressure control and hypertension. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for the rigorous control of expression of the gene encoding 11 beta HSD2, HSD11B2. CpG islands covering the promoter and exon 1 of HSD11B2 were found to be densely methylated in tissues and cell lines with low expression but not those with high expression of HSD11B2. Demethylation induced by 5-aza-2'-deoxycytidine and procainamide enhanced the transcription and activity of the 11 beta HSD2 enzyme in human cells in vitro and in rats in vivo. Methylation of HSD11B2 promoter-luciferase constructs decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including those for Sp1/Sp3, Arnt, and nuclear factor 1 (NF1) diminished their DNA-binding activity. Herein NF1 was identified as a strong HSD11B2 stimulatory factor. The effect of NF1 was dependent on the position of CpGs and the combination of CpGs methylated. A methylated-CpG-binding protein complex 1 transcriptional repression interacted directly with the methylated HSD11B2 promoter. These results indicate a role for DNA methylation in HSD11B2 gene repression and suggest an epigenetic mechanism affecting this gene causally linked with hypertension.

Oxidized guanine lesions as modulators of gene transcription. Altered p50 binding affinity and repair shielding by 7,8-dihydro-8-oxo-2'-deoxyguanosine lesions in the NF-kappaB promoter element.

A number of common promoter elements that drive transcription of redox sensitive genes have runs of guanines in their transcription factor recognition sequence. A paradox exists insomuch that the same guanine runs necessary for transcription factor recognition are thermodynamically prone to oxidative modification, potentially altering the binding affinity of transcription factors. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) is a common oxidative modification of guanine that is generated by a variety of metals and reactive oxygen species. We have used the p50 subunit of the NF-kappaB transcription factor to show that oxidation of guanine to 8-oxo-dG at sites critical for protein recognition impacts transcription factor binding affinity differently depending upon the site of oxidation. It can be argued that the impact of such oxidation will be minimal in repair proficient cells. Therefore, we have developed an assay to assess the ability of these lesions to be shielded by transcription factor binding from recognition and repair by base excision repair (BER) enzymes. In this study, 8-oxo-dG was substituted for guanine at sites G(1)-G(4) in the NF-kappaB sequence 5'-d(AGTTGAG(1)G(2)G(3)G(4)ACTTTCCCAGCC)-3'. We have observed that substitution of 8-oxo-dG at the G(1) site increases p50 binding affinity by approximately 2.5-fold compared to that of the unmodified DNA sequence, while substitution at G(3) reduces the binding affinity by approximately 4-fold. Substitution of 8-oxo-dG at the G(2) and G(4) sites had a minimal impact on p50 binding affinity. Both Escherichia coli fapy glycosylase (Fpg) and human 8-oxo-DNA glycosylase (hOGG1) recognized and cleaved 8-oxo-dG at all four sites within the promoter element. The addition of the p50 transcription factor shielded these lesions from cleavage by the glycosylase in a manner that correlated with the binding affinities of p50 for the different modified sites. These data imply that lesion formation in DNA response elements can modulate gene transcription during oxidative events and that protein binding to these modified sites may allow these lesions to persist on a time scale that impacts global cellular gene transcription.