Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif.

The type I and II scavenger receptors (SRs) are highly restricted to cells of monocyte origin and become maximally expressed during the process of monocyte-to-macrophage differentiation. In this report, we present evidence that SR genomic sequences from -245 to +46 bp relative to the major transcriptional start site were sufficient to confer preferential expression of a reporter gene to cells of monocyte and macrophage origin. This profile of expression resulted from the combinatorial actions of multiple positive and negative regulatory elements. Positive transcriptional control was primarily determined by two elements, located 181 and 46 bp upstream of the major transcriptional start site. Transcriptional control via the -181 element was mediated by PU.1/Spi-1, a macrophage and B-cell-specific transcription factor that is a member of the ets domain gene family. Intriguingly, the -181 element represented a relatively low-affinity binding site for Spi-B, a closely related member of the ets domain family that has been shown to bind with relatively high affinity to other PU.1/Spi-1 binding sites. These observations support the idea that PU.1/Spi-1 and Spi-B regulate overlapping but nonidentical sets of genes. The -46 element represented a composite binding site for a distinct set of ets domain proteins that were preferentially expressed in monocyte and macrophage cell lines and that formed ternary complexes with members of the AP-1 gene family. In concert, these observations suggest a model for how interactions between cell-specific and more generally expressed transcription factors function to dictate the appropriate temporal and cell-specific patterns of SR expression during the process of macrophage differentiation.

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Characterization of PmfR, the Transcriptional Activator of the pAO1-Borne purU-mabO-folD Operon of Arthrobacter nicotinovorans

Journal of Bacteriology ◽

10.1128/jb.187.9.3062-3070.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3062-3070 ◽

Cited By ~ 14

Author(s):

Calin B. Chiribau ◽

Cristinel Sandu ◽

Gabor L. Igloi ◽

Roderich Brandsch

Keyword(s):

Binding Site ◽

Transcriptional Start Site ◽

Transcriptional Activator ◽

Open Reading Frames ◽

Start Site ◽

Gene Encoding ◽

Chloramphenicol Resistance ◽

Nuclease Digestion ◽

Arthrobacter Nicotinovorans

ABSTRACT Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation γ-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating γ-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between −48 and −88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the −35 promoter region of the purU-mabO-folD operon.

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Regulatory elements in the 5'-flanking region and the first intron contribute to transcriptional control of the mouse alpha 1 type I collagen gene

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2224-2227.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2224-2227

Author(s):

R A Rippe ◽

S I Lorenzen ◽

D A Brenner ◽

M Breindl

Keyword(s):

Transcriptional Control ◽

Type I Collagen ◽

Regulatory Elements ◽

Regulatory Sequences ◽

Type I ◽

Col1a1 Gene ◽

First Intron ◽

Flanking Region ◽

Specific Regulation ◽

High Level

We have identified two blocks of regulatory sequences located in the 5'-flanking region and the first intron of the mouse alpha 1 type I collagen (COL1A1) gene. Both blocks were found to contain positive as well as negative regulatory elements. Sequences located within 222 base pairs upstream of the transcription start site showed a strong stimulatory effect on the COL1A1 promoter and were sufficient for tissue-specific regulation of the COL1A1 gene. The combined upstream and intron regulatory sequences showed a marked inhibition of COL1A1 promoter activity in fibroblasts. This finding suggests that additional, more remote regulatory sequences may be required for establishing the high level of activity of the endogenous COL1A1 gene in fibroblastoid cells.

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Transcriptional Regulation ofFucosyltransferase 1Gene Expression in Colon Cancer Cells

The Scientific World JOURNAL ◽

10.1155/2013/105464 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 9

Author(s):

Fumiko Taniuchi ◽

Koji Higai ◽

Tomomi Tanaka ◽

Yutaro Azuma ◽

Kojiro Matsumoto

Keyword(s):

Gene Expression ◽

Colon Cancer ◽

Transcriptional Regulation ◽

Binding Site ◽

Transcriptional Start Site ◽

Dominant Negative ◽

Site Directed Mutagenesis ◽

Luciferase Assay ◽

Start Site ◽

Lewis Y

Theα1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses theFUT1gene, and showsα1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay,FUT1gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation ofFUT1gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression ofFUT1is regulated by Elk-1 in DLD-1 cells.

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Combinatorial interactions between AP-1 and ets domain proteins contribute to the developmental regulation of the macrophage scavenger receptor gene.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2129 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2129-2139 ◽

Cited By ~ 88

Author(s):

H Wu ◽

K Moulton ◽

A Horvai ◽

S Parik ◽

C K Glass

Keyword(s):

Transcription Factors ◽

Scavenger Receptor ◽

Leukemia Cell ◽

Regulatory Elements ◽

Leukemia Cell Line ◽

Macrophage Differentiation ◽

Macrophage Scavenger Receptor ◽

Tetradecanoyl Phorbol Acetate ◽

Ets Domain ◽

Sr Gene

Macrophage development is regulated by a complex set of hormone-like molecules and cell adhesion events that control the growth and differentiation of progenitor cells. The macrophage scavenger receptor (SR) gene becomes markedly upregulated during the final stages of monocyte-to-macrophage differentiation and provides a model for the identification and characterization of transcription factors that control this process. In this report, we have identified three genomic regulatory elements that are required for transactivation of the SR gene in the THP-1 monocytic leukemia cell line following induction of macrophage differentiation by tetradecanoyl phorbol acetate. Each of these regulatory elements contains a near-consensus binding site for members of the AP-1 gene family, while the two most quantitatively important elements also contain juxtaposed binding sites for ets domain transcription factors. We demonstrate that tetradecanoyl phorbol acetate treatment results in a marked and prolonged increase in AP-1 binding activity on these elements, which can be accounted for almost entirely by c-jun and junB. These proteins in turn form ternary complexes with additional factors that bind to the adjacent ets recognition motifs. Several indirect lines of evidence indicate that ets2 represents a component of this ternary complex. The combined expression of c-jun, ets2, and a constitutive form of ras result in synergistic increases in transcription from promoters containing the SR regulatory elements. These observations suggest that SR gene expression is regulated via a signal transduction pathway involving ras, AP-1, and ets domain proteins and imply that at least some of the signalling components involved in ras-dependent growth are also utilized to promote the expression of genes involved in terminal differentiation.

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Identification and transcriptional regulation of the mir-218-1 alternative promoter

10.1101/2020.09.29.319202 ◽

2020 ◽

Author(s):

Brenna A. Rheinheimer ◽

Lukas Vrba ◽

Bernard W Futscher ◽

Ronald L Heimark

Keyword(s):

Transcriptional Regulation ◽

Pancreatic Ductal Adenocarcinoma ◽

Chromatin Immunoprecipitation ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Alternative Promoter ◽

Ductal Adenocarcinoma ◽

Start Site ◽

Public Datasets ◽

Intron 4

AbstractBackgroundmiRNAs are small, endogenous non-coding RNAs approximately 22 nucleotides in length that account for approximately 1% of the genome and play key regulatory roles in multiple signaling pathways. mir-218-1 is an intronic miRNA located within intron 15 of the SLIT2 gene. Public datasets showed enrichment of H3K4me3 within intron 4 of the SLIT2 gene. Therefore, we sought to determine the genomic location and transcriptional regulatory elements of the mir-218-1 candidate alternative promoter in pancreatic ductal adenocarcinoma.MethodsExpression of mir-218 was evaluated in a panel of pancreatic ductal adenocarcinoma cell lines. The mir-218-1 candidate alternative promoter was characterized by chromatin immunoprecipitation, Sequenom, and luciferase assays. Transcriptional regulation of the mir-218-1 candidate alternative promoter was assessed using chromatin immunoprecipitation and an inhibitor to NF-kB.ResultsWe found that expression of mir-218-1 does not correlate with SLIT2 expression and that mir-218-1 has a novel transcriptional start site separate from the SLIT2 promoter. This novel transcriptional start site showed transcriptional activity and was regulated by NF-kB.Conclusionsmir-218-1 is transcribed from an independent and novel transcriptional start site located within intron 4 of the SLIT2 gene in pancreatic ductal adenocarcinoma. Additionally, mir-218-1 expression is regulated by Nf-kB at this alternative transcriptional start site in pancreatic cancer.

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Characterization of the promoter of human adipocyte hormone-sensitive lipase

Biochemical Journal ◽

10.1042/bj3280453 ◽

1997 ◽

Vol 328 (2) ◽

pp. 453-461 ◽

Cited By ~ 30

Author(s):

Jacques GROBER ◽

Henrik LAURELL ◽

Régis BLAISE ◽

Béatrice FABRY ◽

Stéphane SCHAAK ◽

...

Keyword(s):

Genomic Sequence ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Rnase H ◽

Hormone Sensitive Lipase ◽

Start Codon ◽

Start Site ◽

Rnase Protection ◽

Human Adipocyte ◽

Hormone Sensitive

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5ʹ untranslated region (5ʹ-UTR). A single 5ʹ-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5ʹ-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5ʹ-UTR is mutually exclusive. The short and long 5ʹ-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5ʹ-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

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Regulatory elements in the 5'-flanking region and the first intron contribute to transcriptional control of the mouse alpha 1 type I collagen gene.

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2224 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2224-2227 ◽

Cited By ~ 72

Author(s):

R A Rippe ◽

S I Lorenzen ◽

D A Brenner ◽

M Breindl

Keyword(s):

Transcriptional Control ◽

Type I Collagen ◽

Regulatory Elements ◽

Regulatory Sequences ◽

Type I ◽

Col1a1 Gene ◽

First Intron ◽

Flanking Region ◽

Specific Regulation ◽

High Level

We have identified two blocks of regulatory sequences located in the 5'-flanking region and the first intron of the mouse alpha 1 type I collagen (COL1A1) gene. Both blocks were found to contain positive as well as negative regulatory elements. Sequences located within 222 base pairs upstream of the transcription start site showed a strong stimulatory effect on the COL1A1 promoter and were sufficient for tissue-specific regulation of the COL1A1 gene. The combined upstream and intron regulatory sequences showed a marked inhibition of COL1A1 promoter activity in fibroblasts. This finding suggests that additional, more remote regulatory sequences may be required for establishing the high level of activity of the endogenous COL1A1 gene in fibroblastoid cells.

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Transcriptional activation of vesicular monoamine transporter 2 in the pre-B cell line Ea3.123

Biochemical Journal ◽

10.1042/bj3370193 ◽

1999 ◽

Vol 337 (2) ◽

pp. 193-199 ◽

Cited By ~ 9

Author(s):

Fiona WATSON ◽

Damian G. DEAVALL ◽

Janet A. MACRO ◽

Rachel KIERNAN ◽

Rod DIMALINE

Keyword(s):

B Cell ◽

Cell Line ◽

Transcriptional Activation ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Vesicular Monoamine Transporter ◽

Start Site ◽

Monoamine Transporter ◽

Vesicular Monoamine Transporter 2

Uptake and storage of monoamines in secretory granules is accomplished by vesicular monoamine transporters, and it is likely that vesicular monoamine transporter 2 (VMAT2) is important for histamine transport in vivo. In the present study we have used the pre-B-cell line Ea3.123 to investigate the mechanisms involved in the transcriptional activation of the VMAT2 gene. In Ea3.123 cells, VMAT2 mRNA abundance was increased following mobilization of intracellular calcium, and this increased mRNA expression was paralleled by changes in l-histidine decarboxylase mRNA, suggesting that VMAT2 may be responsible for sequestration of histamine into secretory vesicles in this cell line. We cloned the 5´-flanking region of the VMAT2 gene and determined its transcriptional start site by primer extension of rat VMAT2 mRNA. There was no TATA or TATA-like sequence upstream of this region; instead there were GC-rich elements, Ca2+/cAMP-response-element- and SP1-binding motifs. Approx. 900 bp upstream of the transcriptional start site was a purine–pyrimidine repeat sequence that may form a Z-DNA structure. A series of 5´-deletional VMAT2-promoter segments cloned upstream of a luciferase reporter were capable of driving transcription and indicated the presence of multiple regulatory elements, while stimulation with ionomycin or PMA resulted in an increased level of the transcriptional activity of the 5´-promoter segments studied.

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Negative and positive cis-acting elements control the expression of murine alpha 1-protease inhibitor genes.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2625 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2625-2637 ◽

Cited By ~ 10

Author(s):

K T Montgomery ◽

J Tardiff ◽

L M Reid ◽

K S Krauter

Keyword(s):

Protease Inhibitor ◽

Gene Family ◽

Human Gene ◽

Regulatory Elements ◽

Mouse Gene ◽

Start Site ◽

Base Pairs ◽

Negative Element ◽

Cis Acting ◽

Alpha 1 Antitrypsin

The alpha 1-protease inhibitor (alpha 1-PI) proteins of mice are encoded by a group of genes whose members are expressed coordinately in a liver-abundant pattern and are regulated primarily at the transcriptional level. To better understand the developmental and tissue-specific regulation of this gene family, one member that is analogous to the human alpha 1-antitrypsin gene was chosen for study. Deletional analysis of the upstream regulatory region of this gene was performed, spanning from -10 kilobases to -80 base pairs relative to the transcriptional start site. Two functional positive cis-acting elements within the 522 bases immediately upstream of the start site for transcription were shown to modulate the level of expression from this promoter when introduced into human or mouse hepatoma cells, and a third region acted as a negative regulatory element in that its deletion resulted in a two- to sixfold increase of expression of a transfected minigene construct. Sequence comparison between the regulatory domains of two mouse alpha 1-PI genes and the human alpha 1-antitrypsin gene showed that the mouse gene contains a novel positive cis-acting element which is absent in human gene and that a specific eight-base-pair difference between species results in a strong positive cis-acting element in the human gene acting as a negative element in the mouse gene. An enhancer located approximately 3,000 base pairs upstream of the major start site for transcription was also identified. This element is position and orientation independent. Several different DNA-protein binding assays were used to demonstrate that each DNA segment with functional significance in transfection assays interacts specifically with proteins found in adult mouse liver nuclei. The major positive-acting element appeared to be specifically recognized by nuclear proteins found only in tissues that express alpha 1-PI, while the negative element binding proteins were ubiquitous. Thus, the distal regulatory domain including bases -3500 to -133 of this murine alpha 1-PI gene family member is more complex than was previously demonstrated. It is composed of a set of at least three additional functional cis-acting regulatory elements besides those which have been mapped by others and has a far upstream enhancer.

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