Cis-acting Transcriptional Regulatory Elements Research Articles

A genome-wide assessment of conserved SNP alleles reveals a panel of regulatory SNPs relevant to the peripheral nerve

BackgroundIdentifying functional non-coding variation is critical for defining the genetic contributions to human disease. While single-nucleotide polymorphisms (SNPs) within cis-acting transcriptional regulatory elements have been implicated in disease pathogenesis, not all cell types have been assessed and functional validations have been limited. In particular, the cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. We developed a computational pipeline to identify SNPs that affect regulatory function (rSNPs) and evaluated our predictions on a set of 144 regions in Schwann cells, motor neurons, and muscle cells.ResultsWe identified 28 regions that display regulatory activity in at least one cell type and 13 SNPs that affect regulatory function. We then tailored our pipeline to one peripheral nerve cell type by incorporating SOX10 ChIP-Seq data; SOX10 is essential for Schwann cells. We prioritized 22 putative SOX10 response elements harboring a SNP and rapidly validated two rSNPs. We then selected one of these elements for further characterization to assess the biological relevance of our approach. Deletion of the element from the genome of cultured Schwann cells—followed by differential gene expression studies—revealed Tubb2b as a candidate target gene. Studying the enhancer in developing mouse embryos revealed activity in SOX10-positive cells including the dorsal root ganglia and melanoblasts.ConclusionsOur efforts provide insight into the utility of employing strict conservation for rSNP discovery. This strategy, combined with functional analyses, can yield candidate target genes. In support of this, our efforts suggest that investigating the role of Tubb2b in SOX10-positive cells may reveal novel biology within these cell populations.

Interleukin-1 Receptor-Associated Kinase 2- and Protein Kinase D1-Dependent Regulation of IRAK-Monocyte Expression by CpG DNA

As a part of the negative feedback mechanism, CpG DNA induces IRAK-M expression in monocytic cells. In the present study we investigated a biochemical signaling pathway and the transcription factors responsible for CpG DNA-mediated Irak-m gene expression. CpG DNA-induced Irak-m expression did not require new protein synthesis and was regulated at the transcriptional level through an endosomal pH-sensitive TLR9/MyD88 signaling pathway. Over-expression of the dominant negative (DN) form of or gene-specific knockdown of signaling modulators in the TLR9 pathway demonstrated that IRAK4, IRAK1, IRAK2, and PKD1 are required for Irak-m transcription induced by CpG DNA. Over-expression of DN-IRAK1 only partially, but significantly, inhibited CpG DNA-induced Irak-m promoter activity. While IRAK1 was critical for the initial phase, IRAK2 was required for the late phase of TLR9 signaling by sustaining activation of PKD1 that leads to activation of NF-κB and MAPKs. Irak-m promoter-luciferase reporters with alterations in the predicted cis-acting transcriptional regulatory elements revealed that the NF-κB consensus site in the Irak-m promoter region is absolutely required for Irak-m gene expression. AP-1 and CREB binding sites also contributed to the optimal Irak-m expression by CpG DNA. Collectively, our results demonstrate that IRAK2 plays a key role in the TLR9-mediated transcriptional regulation of Irak-m expression by sustaining activation of PKD1 and NF-κB.

Choristoneura fumiferana Granulovirus pk-1: A Baculoviral Protein Kinase

Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk- 1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80 % amino acid identity.

Biallelic transcriptional activation of oncogenic transcription factors in T-cell acute lymphoblastic leukemia

AbstractAberrant expression of transcription factor oncogenes such as HOX11, HOX11L2, TAL1/SCL, LYL1, LMO1, and LMO2 can be detected in lymphoblasts from up to 80% of patients with acute T-cell lymphoblastic leukemia (T-ALL). Transcriptional activation of these oncogenes in leukemic cells typically results from chromosomal rearrangements that place them next to highly active cis-acting transcriptional regulatory elements. However, biallelic activation of TAL1 in some T-ALL cases has been previously proposed. We have used allele-specific mRNA analysis to show that trans-acting mechanisms leading to biallelic overexpression of TAL1 are involved in 10 (42%) of 24 TAL1+ informative T-ALL cases, 2 (17%) of 12 HOX11+ informative cases, and 7 (64%) of 11 LMO2+ informative cases. We propose that aberrant expression of oncogenic transcription factors in a significant fraction of T-ALLs may result from loss of the upstream transcriptional mechanisms that normally down-regulate the expression of these oncogenes during T-cell development.

Myeloid Differentiation Factor 88-dependent Transcriptional Regulation of Cyclooxygenase-2 Expression by CpG DNA: ROLE OF NF-κB AND p38

CpG DNA induces macrophage cyclooxgenase-2 (Cox-2) production. In this study, we have investigated a biochemical signaling pathway and transcription factors responsible for transcriptional regulation of the Cox-2 gene expression induced by CpG DNA. CpG DNA-induced Cox-2 promoter activity was completely inhibited by an endosomal acidification inhibitor (chloroquine), a TLR9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. In contrast, overexpression of DN-IRAK1 or DN-TRAF6 only partially inhibited CpG DNA-induced Cox-2 promoter activity and NF-kappaB activation, indicating the presence of additional signaling modulators downstream of MyD88. CpG DNA-induced Cox-2 promoter activity was substantially suppressed in cells overexpressing super-suppressive IkappaB (IkappaB-arachidonic acid), DN-p38, or DN-CREB. In addition, Cox-2 promoter-luciferase reporters with alterations in predicted cis-acting transcriptional regulatory elements revealed that C/EBP, Ets-1, NF-kappaB, and CREB-binding sites are essential for optimal Cox-2 expression in response to CpG DNA. Conclusively, these results demonstrate that endosomal DNA processing and TLR9/MyD88-dependent activation of NF-kappaB and p38 are required for transcriptional regulation of Cox-2 expression induced by CpG DNA, and suggest that interleukin-1 receptor-associated kinase and/or TRAF6 may be a diverging point for NF-kappaB activation in response to CpG DNA in RAW264.7 cells.

Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice.

The pulmonary endothelin (ET) system has been implicated in the pathogenesis of chronic lung diseases such as pulmonary hypertension, asthma, chronic obstructive lung disease, idiopathic pulmonary fibrosis, and bronchiolitis obliterans. However, the etiologic role of ET-1 in these diseases has not yet been established. We recently demonstrated that ET-1 transgenic mice, generated using the human prepro-ET-1 expression cassette including the cis-acting transcriptional regulatory elements, had predominant transgene expression in lung, brain, and kidney. We used these mice in the present study to analyze the pathophysiologic consequences of long-term pulmonary overexpression of ET-1. We found that ET-1 overexpression in the lungs did not result in significant pulmonary hypertension, but did result in development of a progressive pulmonary fibrosis and recruitment of inflammatory cells (predominantly CD4-positive cells). Our study provides evidence that a long-term activated pulmonary ET system, without any other stimuli, produces chronic lymphocytic inflammation and lung fibrosis. This suggests that overexpression of ET-1 may be a central event in the pathogenesis of lung diseases associated with fibrosis and chronic inflammation, such as pulmonary fibrosis and bronchiolitis.

Transcriptional Activation of the Cyclooxygenase-2 Gene in Endotoxin-treated RAW 264.7 Macrophages

Cyclooxygenase-2 (COX-2), the enzyme primarily responsible for induced prostaglandin synthesis, is an immediate early gene induced by endotoxin in macrophages. We investigated the cis-acting elements of the COX-2 5'-flanking sequence, the transcription factors and signaling pathways responsible for transcriptional activation of the COX-2 gene in endotoxin-treated murine RAW 264.7 macrophages. Luciferase reporter constructs with alterations in presumptive cis-acting transcriptional regulatory elements demonstrate that the cyclic AMP-response element and two nuclear factor interleukin-6 (CCAAT/enhancer-binding protein (C/EBP)) sites of the COX-2 promoter are required for optimal endotoxin-dependent induction. In contrast, the E-box and NF-kappaB sites are not required for endotoxin-dependent induction. Inhibition of endotoxin-induced NF-kappaB activation by expression of an inhibitor-kappaB alpha mutant does not block endotoxin-dependent COX-2 reporter activity. Overexpression of c-Jun, C/EBPbeta, and C/EBPdelta enhances induction of the COX-2 reporter, while overexpression of cyclic AMP-response element-binding protein or "dominant negative" C/EBPbeta represses COX-2 induction. In addition, endotoxin rapidly and transiently elicits c-Jun phosphorylation in RAW 264.7 macrophages. Cotransfection of the COX-2 reporter with dominant negative expression vectors shows that endotoxin-induced COX-2 gene expression requires signaling through a Ras-independent pathway involving the adapter protein ECSIT and the signaling kinases MEKK1 and JNK. In contrast, endotoxin-induced COX-2 reporter activity is not blocked by overexpression of dominant-negative forms of Raf-1, ERK1, or ERK2.

Identification and characterization of cis-acting regulatory elements for the expression of the human aromatase cytochrome P-450 gene.

CYP19, the human aromatase cytochrome P-450 (P450arom) gene, encodes an enzyme which converts androgens to estrogens by three successive hydroxylation reactions by coupling with NADPH-cytochrome P-450 reductase. In the present study, we have characterized two cis-acting transcriptional regulatory elements of CYP19, termed as hATRE-1 (human aromatase cytochrome P-450 gene transcriptional regulatory element-1) ([sequence: see text]) and hATRE-2 ([sequence: see text]). These sequences are located between -2238 and -2214, and between -2141 and -2098 relative to the major cap site of the gene, respectively. Transient expression analysis in human BeWo choriocarcinoma cells, in which CYP19 is expressed, shows that hATRE-1 represses the expression of the bacterial chloramphenicol acetyltransferase reporter gene driven by the promoter of CYP19, whereas hATRE-2 enhances the reporter gene expression in response to 12-O-tetradecanoylphorbol 13-acetate. Electrophoretic mobility shift analysis indicates that nuclear binding factors specific to hATRE-1 are present in BeWo cells, but not in HeLa cells nor in TYK-nu cells that lack the expression of CYP19. In contrast, nuclear binding factors to hATRE-2 are present not only in BeWo cells but also in the latter two types of cells. Nevertheless, hATRE-2 does not affect the reporter gene expression in HeLa cells and TYK-nu cells. These results indicate that hATRE-1 and hATRE-2 are cis-acting transcriptional regulatory elements involving in the regulation of the cell type-specific expression of CYP19.

A complex response element in intron 1 of the androgen-regulated 20-kDa protein gene displays cell type-dependent androgen receptor specificity.

The androgen-regulated 20-kDa protein gene consists of four exons that code for a major secretory protein of rat ventral prostate. Analysis of its potential cis-acting transcriptional regulatory elements revealed that a large intron 1 region (In-1) had stronger androgen response element (ARE) activity than did the 5'-flanking DNA. In cotransfected CV1 cells, In-1 and its most active subfragment In-1c functioned as AREs but not glucocorticoid response elements (GRE). Nevertheless several ARE/GRE-like partial palindromic sequences are present in In-1c, and it bound both androgen receptors and glucocorticoid receptors in mobility shift assays. A cluster of three ARE/GRE-like sequences contained within a 39-base pair sequence of In-1c had both ARE and GRE activities when analyzed as an isolated oligonucleotide, suggesting that other elements within In-1c determined its ARE specificity. In addition to ARE/GRE-like sequences, In-1c contains putative response elements for the transcription factors AP1, CREB, AP2, OCT-1, C/EBP, and a number of inverted and direct repeats. The ARE specificity of In-1c observed in CV1 cells was diminished in PC3 and HeLa cells transiently cotransfected with an androgen receptor or glucocorticoid receptor expression vector together with an In-1c reporter vector; however, the ARE activity of In-1c was greater than its GRE activity in these cell lines. Interestingly, a 131-base pair subfragment of In-1c retained ARE specificity in all three cell lines.

5' distal and proximal cis-acting regulator elements are required for developmental control of a rice seed storage protein glutelin gene.

Using a homologous transgenic rice system it is demonstrated that 5' distal and proximal cis-acting transcriptional regulatory elements are required for developmental control of a rice seed storage protein glutelin gene. Analyses of gene expression of nine progressively truncated 5' promoter sequences in developing endosperm indicated the existence of at least one major positive element located from the -5.1 to -1.8 kb region. The functional importance of proximal elements in the context of 1.8 kb promoter was demonstrated by single substitution mutations in the TATA box (-28/-23), AACA motif (-73/-61), and protein-binding boxes I (-103/-86), II (-124/-110), III (-175/-158) and IV (-200/-217). A simultaneous mutation of five protein-binding sites (-410/-86) essentially eliminated the activity of the 1.8 kb promoter. Although temporal control of the Gt1 gene during endosperm development was retained in plants of constructs from -5.1 kb to -155 bp, spatial control of the glutelin gene was altered when the 5.1 kb promoter was deleted to -507 bp or -154 bp as the reporter gene activities of these constructs were detected in phloem of leaves, and in stems, sheaths and roots of plants.

Identification of a nuclear factor that binds to a conserved sequence of the I-A beta gene.

Human and murine class II genes of the MHC show a striking homology 50 to 120 bp upstream of the transcription start site. This area is composed of two conserved sequences (a 13-mer and an 8-mer separated by 19 to 20 bp). Recently, these conserved sequences have been identified as cis-acting transcriptional regulatory elements. We have sought nuclear factors that bind specifically to an upstream fragment (-245 to +75 bp) of the murine I-A beta chain gene that contains the conserved sequences by application of a modified gel electrophoresis DNA binding assay. We report here the identification of a nuclear factor whose binding site overlaps the 8-mer conserved sequence. This factor is present in murine B and T lymphocytes, macrophages, mastocytes, fibroblasts, and human B lymphocytes and macrophages. The binding site was defined by using DNase I and dimethylsulfate protection assays. The putative binding sequence is closely related to the sequence, CCAAT, which is often found associated with the promoter of a gene and is recognized by the transcriptional factors CCAAT-binding transcription factor and nuclear factor I. Oligonucleotides that contain the binding site sequences for nuclear factor I and the alpha-globin CCAAT element, however, do not compete for binding of the nuclear factor to the sequence identified here, suggesting that, in spite of the similarity of the binding sequence, the nuclear factor identified in this report may be different. This nuclear protein may be one of the trans-acting factors that mediate transcription of class II genes.

A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes.

Trans-acting factors that mediate B-cell specific transcription of immunoglobulin genes have been postulated based on an analysis of the expression of exogenously introduced immunoglobulin gene recombinants in lymphoid and non-lymphoid cells. Two B-cell-specific, cis-acting transcriptional regulatory elements have been identified. One element is located in the intron between the variable (V) and constant (C) regions of both heavy and kappa light-chain genes and acts as a transcriptional enhancer. The second element is found upstream of both heavy and kappa light-chain gene promoters. This element directs lymphoid-specific transcription even in the presence of viral enhancers. We have sought nuclear factors that might bind specifically to these two regulatory elements by application of a modified gel electrophoresis DNA binding assay. We report here the identification of a human B-cell nuclear factor (IgNF-A) that binds to DNA sequences in the upstream regions of both the mouse heavy and kappa light-chain gene promoters and also to the mouse heavy-chain gene enhancer. This sequence-specific binding is probably mediated by a highly conserved sequence motif, ATTTGCAT, present in all three transcriptional elements. Interestingly, a factor showing similar binding specificity to IgNF-A is also present in human HeLa cells.