The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation

1989 ◽  
Vol 9 (11) ◽  
pp. 4759-4766
Author(s):  
F Tronche ◽  
A Rollier ◽  
I Bach ◽  
M C Weiss ◽  
M Yaniv
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Transient Transfection ◽  
Tata Box ◽  
Specific Factor ◽  
Target Sequence ◽  
Functional Interaction ◽  
Dna Methylase ◽  
Ccaat Box

We have characterized in the accompanying paper (P. Herbomel, A. Rollier, F. Tronche, M.-O. Ott, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 9:4750-4758, 1989) six different elements in the albumin promoter. One of them, the proximal element (PE), is the binding site for a strictly liver specific factor, APF/HNF1. This binding site contains a bacterial DAM DNA methylase methylation target sequence which, when methylated, decreases the affinity of the protein for this element. When the different albumin promoter constructions were prepared in an Escherichia coli deoxyadenosine methylase-negative strain, the respective contributions of the elements to the overall promoter activity were strikingly different. An intact proximal element plus the TATA box gave almost full transcriptional activity in transient transfection experiments and only in differentiated hepatoma cells of line H4II, whereas the distal elements (distal element III [DEIII], the NF1-binding site DEII, and the E/CBP-binding site DEI) had become essentially dispensable. Mutations affecting the CCAAT box showed only a two- to threefold decrease. When PE was methylated, mutated, or replaced by the homologous element from the alpha-fetoprotein gene, activity in the context of the short promoter (PE plus the TATA box) was abolished. However, activity was restored in the presence of the upstream elements, showing that cooperation with factors binding to the CCAAT box and distal elements favors the functional interaction of the liver-specific APF/HNF1 factor with lower-affinity binding sites.

scholarly journals The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation.

1989 ◽  
Vol 9 (11) ◽  
pp. 4759-4766 ◽  
Author(s):  
F Tronche ◽  
A Rollier ◽  
I Bach ◽  
M C Weiss ◽  
M Yaniv
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Transient Transfection ◽  
Tata Box ◽  
Specific Factor ◽  
Target Sequence ◽  
Functional Interaction ◽  
Dna Methylase ◽  
Ccaat Box

We have characterized in the accompanying paper (P. Herbomel, A. Rollier, F. Tronche, M.-O. Ott, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 9:4750-4758, 1989) six different elements in the albumin promoter. One of them, the proximal element (PE), is the binding site for a strictly liver specific factor, APF/HNF1. This binding site contains a bacterial DAM DNA methylase methylation target sequence which, when methylated, decreases the affinity of the protein for this element. When the different albumin promoter constructions were prepared in an Escherichia coli deoxyadenosine methylase-negative strain, the respective contributions of the elements to the overall promoter activity were strikingly different. An intact proximal element plus the TATA box gave almost full transcriptional activity in transient transfection experiments and only in differentiated hepatoma cells of line H4II, whereas the distal elements (distal element III [DEIII], the NF1-binding site DEII, and the E/CBP-binding site DEI) had become essentially dispensable. Mutations affecting the CCAAT box showed only a two- to threefold decrease. When PE was methylated, mutated, or replaced by the homologous element from the alpha-fetoprotein gene, activity in the context of the short promoter (PE plus the TATA box) was abolished. However, activity was restored in the presence of the upstream elements, showing that cooperation with factors binding to the CCAAT box and distal elements favors the functional interaction of the liver-specific APF/HNF1 factor with lower-affinity binding sites.

Overexpression of CCAAT Displacement Protein Represses the Promiscuously Active Proximal gp91phox Promoter

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3151-3160 ◽  
Author(s):  
Diana Catt ◽  
Shannon Hawkins ◽  
Ann Roman ◽  
Wen Luo ◽  
David G. Skalnik
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Binding Activity ◽  
Proximal Promoter ◽  
Modular Organization ◽  
Transcriptional Activators ◽  
Cis Element ◽  
Dna Binding Activity ◽  
Ccaat Displacement Protein

CCAAT displacement protein (CDP) is a transcriptional repressor that restricts expression of the gp91phox gene to mature myeloid cells. CDP interacts with multiple sites within the −450 to +12 bp human gp91phox promoter, and down-regulation of CDP DNA-binding activity is required for induction of gp91phox transcription in mature phagocytes. Truncation of the gp91phox promoter to −102 to +12 bp removes 4 CDP-binding sites and reveals a promiscuous promoter activity that is active in some nonphagocytic cells. A cis-element at −90 bp is required for derepressed transcription and serves as a binding site for multiple transcriptional activators. We now report that this element also serves as a binding site for CDP. The affinity of CDP for this element is relatively weak compared with upstream CDP-binding sites within the promoter, consistent with the promiscuous transcriptional activity exhibited by the −102 to +12 bp gp91phox promoter fragment. Further analysis of the proximal promoter reveals an additional weak-affinity CDP-binding site centered at approximately −20 bp. Overexpression of cloned CDP represses the −102 to +12 bp gp91phox promoter, indicating that these proximal CDP-binding sites are functionally significant. The constellation of transcriptional activators and a repressor that interacts with the −90 bp cis-element is identical to that observed for a promoter element at −220 bp, reflecting the highly modular organization of the gp91phoxpromoter. These studies illustrate the complex interplay between transcriptional activators and a repressor that contribute to the myeloid-restricted expression of the gp91phox gene.

Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity

1994 ◽  
Vol 14 (11) ◽  
pp. 7592-7603
Author(s):  
P E Kroeger ◽  
R I Morimoto
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Cellular Stress Response ◽  
Specific Factor ◽  
Heat Shock Element ◽  
Cooperative Interactions ◽  
Dna Binding Sites

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

scholarly journals Expression of the woodchuck N-myc2 retroposon in brain and in liver tumors is driven by a cryptic N-myc promoter.

1992 ◽  
Vol 12 (12) ◽  
pp. 5336-5344 ◽  
Author(s):  
G Fourel ◽  
C Transy ◽  
B C Tennant ◽  
M A Buendia
Keyword(s):  
Binding Sites ◽  
Liver Tumors ◽  
Cpg Island ◽  
Evolutionary Conservation ◽  
Transient Transfection ◽  
Tata Box ◽  
Potential Binding ◽  
Myc Gene ◽  
Low Levels ◽  
Different Tissues

The woodchuck intronless proto-oncogene N-myc2 was initially discovered as a frequent target site for hepadnavirus integration in hepatocellular carcinoma. N-myc2 possesses characteristics of a functional retroposon derived from the woodchuck N-myc gene. We have investigated the regulatory signals governing N-myc2 expression and found that a short promoter, including a variant TATA box and potential binding sites for several transcription factors, is localized in the N-myc2 sequences homologous to the 5' untranslated region of the second N-myc exon. The corresponding region in the intron-containing woodchuck N-myc gene also exhibited promoter activity in transient transfection assays. The high evolutionary conservation of these sequences in mammalian N-myc genes suggests that they contain a cryptic N-myc promoter which may be unmasked in the particular context provided by the N-myc2 retroposon. Although N-myc2, like the woodchuck N-myc gene, contributes to an extended CpG island and was found constitutively hypomethylated, it presents a highly restricted expression pattern in adult animals. Whereas the intron-containing N-myc gene is expressed at low levels in different tissues, N-myc2 mRNA was detected only in brain tissue, raising questions about the functional significance of the maintenance of a second N-myc gene in the woodchuck genome.

In vivo stimulation of a chimeric promoter by binding sites for nuclear factor I

1991 ◽  
Vol 11 (6) ◽  
pp. 2946-2951
Author(s):  
J J Knox ◽  
P J Rebstein ◽  
A Manoukian ◽  
R M Gronostajski
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Tata Box ◽  
Nuclear Factor ◽  
Chimeric Promoter ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box.

Liver-specific gene expression: A-activator-binding site, a promoter module present in vitellogenin and acute-phase genes

1991 ◽  
Vol 11 (1) ◽  
pp. 93-101
Author(s):  
M Kaling ◽  
W Kugler ◽  
K Ross ◽  
C Zoidl ◽  
G U Ryffel
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Acute Phase ◽  
Interleukin 6 ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
In Vitro Transcription ◽  
Gel Retardation ◽  
Vitellogenin Gene

The A2 vitellogenin gene of Xenopus laevis, which is expressed liver specifically, contains an A-activator-binding site (AABS) that mediates high in vitro transcriptional activity in rat liver nuclear extracts. Footprint experiments with DNase I and gel retardation assays revealed the binding of several proteins to AABS. Using binding sites of known DNA-binding proteins as competitors in the gel retardation assay, we found that the transcription factor C/EBP and/or one of its "iso-binders" as well as LFB1/HNF1 bound AABS. These interactions were confirmed by in vitro transcription experiments using various oligonucleotides as competitors. However, saturating amounts of C/EBP- and LFB1/HNF1-binding sites as competitors only partially blocked AABS-mediated transcriptional activity. This finding implies that at least a third distinct transcription factor interacts with AABS. In vitro transcription experiments revealed that AABS was present not only in the closely related Xenopus A1 vitellogenin gene but also in acute-phase genes as a liver-specific regulatory element known to confer the interleukin-6 response. Both AABS and the interleukin-6 response element are promoter modules interacting with at least three distinct transcription factors, including C/EBP and LFB1/HNF1.

scholarly journals The Role of Forkhead Box A2 to Restrict Androgen-Regulated Gene Expression of Lipocalin 5 in the Mouse Epididymis

2006 ◽  
Vol 20 (10) ◽  
pp. 2418-2431 ◽  
Author(s):  
Xiuping Yu ◽  
Kichiya Suzuki ◽  
Yongqing Wang ◽  
Aparna Gupta ◽  
Renjie Jin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Binding Site ◽  
Binding Sites ◽  
Transient Transfection ◽  
Specific Gene ◽  
Promoter Fragment ◽  
Specific Gene Expression ◽  
Principal Cells ◽  
Forkhead Box

Abstract Murine epididymal retinoic acid-binding protein [or lipocalin 5 (Lcn5)] is synthesized and secreted by the principal cells of the mouse middle/distal caput epididymidis. A 5-kb promoter fragment of the Lcn5 gene can dictate androgen-dependent and epididymis region-specific gene expression in transgenic mice. Here, we reported that the 1.8-kb Lcn5 promoter confers epididymis region-specific gene expression in transgenic mice. To decipher the mechanism that directs transcription, 14 chimeric constructs that sequentially removed 100 bp of 1.8-kb Lcn5 promoter were generated and transfected into epididymal cells and nonepididymal cells. Transient transfection analysis revealed that 1.3 kb promoter fragment gave the strongest response to androgens. Between the 1.2-kb to 1.3-kb region, two androgen receptor (AR) binding sites were identified. Adjacent to AR binding sites, a Foxa2 [Fox (Forkhead box) subclass A] binding site was confirmed by gel shift assay. Similar Foxa binding sites were also found on the promoters of human and rat Lcn5, indicating the Foxa binding site is conserved among species. We previously reported that among the three members of Foxa family, Foxa1 and Foxa3 were absent in the epididymis whereas Foxa2 was detected in epididymal principal cells. Here, we report that Foxa2 displays a region-specific expression pattern along the epididymis: no staining observed in initial segment, light staining in proximal caput, gradiently heavier staining in middle and distal caput, and strongest staining in corpus and cauda, regions with little or no expression of Lcn5. In transient transfection experiments, Foxa2 expression inhibits AR induction of the Lcn5 promoter, which is consistent with the lack of expression of Lcn5 in the corpus and cauda. We conclude that Foxa2 functions as a repressor that restricts AR regulation of Lcn5 to a segment-specific pattern in the epididymis.

scholarly journals Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity.

1994 ◽  
Vol 14 (11) ◽  
pp. 7592-7603 ◽  
Author(s):  
P E Kroeger ◽  
R I Morimoto
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Cellular Stress Response ◽  
Specific Factor ◽  
Heat Shock Element ◽  
Cooperative Interactions ◽  
Dna Binding Sites

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

C/EBPalpha Is a Key Transcriptional Regulator of the Triggering Receptor on Myeloid Cells-2 Gene

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4762-4762
Author(s):  
Maria Michela Mancarelli ◽  
Lily Catherine Frusciante ◽  
Gabrielle Francoise Cauvi ◽  
Charles De Rossi ◽  
Bruce Edward Torbett
Keyword(s):  
Gene Regulation ◽  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Fold Increase ◽  
Full Length ◽  
And Function

Abstract Triggering receptor on myeloid cells-2 (TREM-2) is a member of the innate immune signaling receptor TREM family. After differentiation of monocytes, TREM-2 is expressed on the cell surface of macrophages, monocyte-derived dendritic cells, microglia, and osteoclasts. TREM-2 is necessary for modulation of cellular activation and function, and loss of TREM-2 results in the decrease of phagocytosis of apoptotic neurons and cells and an increase in inflammation response of macrophage-lineage cells. To better understand TREM-2 gene regulation during monocyte differentiation we isolated and then analyzed the putative human TREM-2 promoter to identify transcriptional control. The 1131 bp TREM- 2 promoter contains binding sites for C/EBPα and PU.1 transcription factors, known for regulation of myeloid cell differentiation and function. A series of 5 promoter reporter deletion mutants were generated to dissect transcription regulation. We progressively deleted the full-length promoter, −1002/+129, of HIF1, STAT5, GATA1, YY1F, C/EBPα binding sites generating −866/+129, −632/+129, −526/+129, −258/+129, +37/+129 promoter reporters, respectively. All TREM-2 promoter reporters showed minimal transcriptional activity when transfected in 293T-HEK cells. In contrast, C/EBPα was necessary and sufficient for TREM-2 promoter reporter activity in co-transfection studies, demonstrating a 5-fold induction of transcriptional activity as compared to controls. Consistent with the TREM-2 reporter findings showing C/EBPα mediated activity, EMSA assays demonstrated specific C/EBPα binding to sites within the TREM-2 promoter. To determine if both C/EBPα binding sites in TREM-2 were necessary for activity we generated loss of C/EBPα binding site reporters and observed that only the −43/−28 C/EBPα binding site was essential for activity. To clarify if +73/+93 PU.1 binding site had a role in TREM-2 regulation, we co-transfected the full-length promoter with PU.1 and C/EBPα and observed a 10-fold increase of induction of TREM-2 transcriptional activity. However, PU.1 alone did not activate the full-length TREM-2 promoter or bind to the PU.1 consensus site, and loss of the PU.1 binding site did not alter transcriptional activity. These results suggest a key role for direct C/EBPα and indirect PU.1 participation in regulation of TREM-2. Studies are underway to precisely understand TREM-2 gene regulation and how TREM-2 controls macrophage-mediated phagocytosis and inflammation.

Overexpression of CCAAT Displacement Protein Represses the Promiscuously Active Proximal gp91phox Promoter

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3151-3160 ◽  
Author(s):  
Diana Catt ◽  
Shannon Hawkins ◽  
Ann Roman ◽  
Wen Luo ◽  
David G. Skalnik
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Binding Activity ◽  
Proximal Promoter ◽  
Modular Organization ◽  
Transcriptional Activators ◽  
Cis Element ◽  
Dna Binding Activity ◽  
Ccaat Displacement Protein

Abstract CCAAT displacement protein (CDP) is a transcriptional repressor that restricts expression of the gp91phox gene to mature myeloid cells. CDP interacts with multiple sites within the −450 to +12 bp human gp91phox promoter, and down-regulation of CDP DNA-binding activity is required for induction of gp91phox transcription in mature phagocytes. Truncation of the gp91phox promoter to −102 to +12 bp removes 4 CDP-binding sites and reveals a promiscuous promoter activity that is active in some nonphagocytic cells. A cis-element at −90 bp is required for derepressed transcription and serves as a binding site for multiple transcriptional activators. We now report that this element also serves as a binding site for CDP. The affinity of CDP for this element is relatively weak compared with upstream CDP-binding sites within the promoter, consistent with the promiscuous transcriptional activity exhibited by the −102 to +12 bp gp91phox promoter fragment. Further analysis of the proximal promoter reveals an additional weak-affinity CDP-binding site centered at approximately −20 bp. Overexpression of cloned CDP represses the −102 to +12 bp gp91phox promoter, indicating that these proximal CDP-binding sites are functionally significant. The constellation of transcriptional activators and a repressor that interacts with the −90 bp cis-element is identical to that observed for a promoter element at −220 bp, reflecting the highly modular organization of the gp91phoxpromoter. These studies illustrate the complex interplay between transcriptional activators and a repressor that contribute to the myeloid-restricted expression of the gp91phox gene.

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