Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5' upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a beta-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.
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PMID:Novel DNA binding proteins participate in the regulation of human neurofilament H gene expression. 127 52

Na,K-ATPase alpha 1 subunit gene (ATP1A1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATP1A1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Sp1 binding site), from -102 to -61, and from -58 to -49 (including an Sp1 consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATP1A1 regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.
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PMID:Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind. 132 13

The ability of the human insulin receptor promoter to direct expression of a linked chloramphenicol acetyltransferase gene was assessed in transient transfections into HepG2 and Hela cells. A 5'-deletional analysis of the promoter showed that regions between -646 and -489 were important for the activity of the proximal promoter. In addition, a possible negative regulatory element was identified between -1311 and -877 and a positive element between -1823 and -1311. DNase I footprint and gel retardation analysis showed that multiple factors bind to the human insulin receptor promoter. In particular, DNase I protection patterns were observed over the Sp1 sites at -620 to -599 and -438 to -392, a TC box at -533, four homopyrimidine/homopurine sites clustered around -1150, and a site at -1420 that contains the motif TGGCCC which has been shown to bind the liver-specific transcription factor LF-A1.
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PMID:Transcriptional regulation of the human insulin receptor promoter. 132 34

The DNA-binding proteins which recognize the regulatory sequence elements of the hepatitis B virus (HBV) major surface antigen promoter were examined by gel retardation analysis, using nuclear extracts from the human hepatoma cell line Huh7. Using this assay, we identified four regions (B, D, E, and F) of the promoter that interact with the same or similar transcription factor(s). In addition, the recognition sequence for the Sp1 transcription factor bound the same or similar transcription factor(s) present in Huh7 cell nuclear extracts, and this binding was inhibited by the four major surface antigen promoter elements, B, D, E, and F. Purified Sp1 transcription factor was shown to bind to three (B, D, and F) of the major surface antigen promoter regulatory sequence elements by DNase I footprinting. Using transient transfection assays with Drosophila Schneider line 2 cells, we found that transcription from the major surface antigen promoter was transactivated by exogenously expressed Sp1, whereas transcription from the other three HBV promoters was not. Deletion analysis of the major surface antigen promoter demonstrated that the promoter region between -35 and +157 was sufficient to confer Sp1 responsiveness. This promoter region includes one of the regulatory elements footprinted by the purified Sp1 transcription factor. The function of the B, D, E, and F promoter elements was further examined by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. This finding demonstrates that the HBV major surface antigen promoter contains four functional Sp1 binding sites which probably contribute to the level of expression from this promoter during viral infection.
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PMID:Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor. 133 2

In order to identify potential regulatory elements of the human mid-sized (M) neurofilament (NF) gene we preformed DNase I footprinting, gel mobility shift assays and methylation interference studies with probes from the NF(M) immediate 5' flanking region. These studies identified multiple sites for DNA-binding proteins including four Sp1 sites, and single sites each for members of the NF-1 and AP-1 families of DNA binding proteins. In addition a binding site within a pyrimidine tract likely binds a novel DNA-binding protein which also interacts with the human NF(H) gene promoter. Factors that bind to these sites are found in both neural and non-neural cells suggesting that the NF(M) promoter may not contain tissue specific regulatory signals. In transient assays, addition of these binding sites to an NF(M) minimal promoter containing only a TATA box lead to a greater than 40-fold activation of transcription over background. Progressive 5' deletions reduced expression in a step wise manner suggesting that all the factors likely act synergistically as positive regulators of transcription.
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PMID:Multiple nuclear factors interact with the promoter of the human neurofilament M gene. 133 73

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.
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PMID:Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase. 133 64

A unique cAMP regulatory sequence, -129/-96 base pairs (bp), associated with the gene encoding human cytochrome P450C21 (CYP21B) binds a nuclear protein designated ASP, as described previously (Kagawa, N., and Waterman, M. R. (1991) J. Biol. Chem. 266, 11199-11204). This putative transcription factor required for cAMP-dependent transcription of the human CYP21B gene has been purified from the nuclear extracts of mouse Y1 cells by using sequence-specific DNA-affinity chromatography. The purified ASP is 78 kDa as estimated by SDS-polyacrylamide gel electrophoresis and binds to its specific recognition site, -126/-113-bp CACTCTGTGGGCGG, which has been demonstrated to be the minimum cAMP regulatory sequence of the human CYP21B gene. To characterize ASP more precisely, an antibody was raised against the 78-kDa protein. This antibody led to a supershift of the DNA.ASP complex on gel shift analysis and inhibition of in vitro transcription promoted by the ASP binding sequence, thereby indicating that ASP is a 78-kDa transcription factor. Upon DNase I footprinting experiments, ASP showed a characteristic footprint which very closely resembles but is distinct from that of Sp1 which also occupies a binding site within -129/-96 bp. Furthermore, the addition of purified ASP enhanced the mRNA synthesis promoted by the minimum cAMP regulatory sequence in a cell-free transcription system using HeLa cell extracts, whereas added Sp1 does not. These results indicate that ASP is a primary transcription factor for the cAMP-dependent regulation of the human CYP21B gene.
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PMID:Purification and characterization of a transcription factor which appears to regulate cAMP responsiveness of the human CYP21B gene. 133 85

Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor.
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PMID:5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression. 138 Jun 48

To explore the role of transcriptional factors in the genesis of the senescent phenotype, nuclear extracts from 4- and 30-month-old rat brains were analyzed for the presence of DNA-binding proteins able to interact with double-stranded oligonucleotides containing recognition sites for sequence-specific DNA-binding factors. Gel shift assays revealed that the DNA-binding efficiency of Sp1 is significantly reduced in aged animals compared to young ones, whereas CTF/NF1 and AP1 from young and old rat nuclear extracts bind their DNA targets with the same efficiency. The quantitative analysis of Sp1 by immunoblotting indicated that equivalent quantities and degrees of heterogeneity of Sp1 protein are present in both nuclear extracts, suggesting that the observed difference is not due to a different expression of this transcriptional factor. DNase I footprinting of the heavy chain ferritin gene promoter, which contains a Sp1 binding site, demonstrated that the nuclear extract from 30-month-old rat brain does not protect the region involved in the regulation of the H ferritin gene by Sp1. This results in a reduction of about 50% of the expression of the H ferritin mRNA in aged rat brains. Furthermore, the Sp1 binding sites present in the SV40 early promoter are not protected in a DNase I footprinting assay where a nuclear extract from 30-month-old rat brain was used as a source of DNA binding proteins. Liver nuclear extracts prepared from young and aged rats demonstrated that a decrease of Sp1 binding efficiency is similarly present in this tissue.
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PMID:Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues. 138 57

In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.
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PMID:Promoter region of the human alpha 2A adrenergic receptor gene. 138 31


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