A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

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Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3002-3014.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3002-3014

Author(s):

K Kudrycki ◽

C Stein-Izsak ◽

C Behn ◽

M Grillo ◽

R Akeson ◽

...

Keyword(s):

Nuclear Protein ◽

Consensus Sequence ◽

Binding Motif ◽

Sequence Motif ◽

Olfactory Neuron ◽

Sequence Motifs ◽

Mobility Shift ◽

Specific Expression ◽

Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

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The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3860-3871.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3860-3871

Author(s):

P L Sawaya ◽

B R Stripp ◽

J A Whitsett ◽

D S Luse

Keyword(s):

Transcription Factors ◽

Rat Liver ◽

Transcription Initiation ◽

Clara Cell ◽

Clara Cells ◽

Mobility Shift ◽

Specific Expression ◽

Fetal Sheep ◽

Region I

We have shown that a large fragment (-2339 to +57) from the rat CC10 gene directed lung-specific expression of a reporter construct in transgenic animals. Upon transfection, a smaller fragment (-165 to +57) supported reporter gene expression exclusively in the Clara cell-like NCI-H441 cell line, suggesting that a Clara cell-specific transcriptional element resided on this fragment (B. R. Stripp, P. L. Sawaya, D. S. Luse, K. A. Wikenheiser, S. E. Wert, J. A. Huffman, D. L. Lattier, G. Singh, S. L. Katyal, and J. A. Whitsett, J. Biol. Chem. 267:14703-14712, 1992). The interactions of nuclear proteins with a particular segment of the CC10 promoter which extends from 79 to 128 bp upstream of the CC10 transcription initiation site (CC10 region I) have now been studied. This sequence can stimulate both in vitro transcription in H441 nuclear extract and transient expression of reporter constructs in H441 cells. Electrophoretic mobility shift assays using extracts from H441, HeLa, rat liver, and fetal sheep lung cells were used to demonstrate that members of the AP-1, octamer, and HNF-3 families bind to CC10 region I. Transcription factors from H441 cells which are capable of binding to CC10 region I are either absent in HeLa, rat liver, and fetal sheep lung extracts or enriched in H441 extracts relative to extracts from non-Clara cells.

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Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01634 ◽

2005 ◽

Vol 34 (1) ◽

pp. 177-197 ◽

Cited By ~ 28

Author(s):

A Cote-Vélez ◽

L Pérez-Martínez ◽

M Y Díaz-Gallardo ◽

C Pérez-Monter ◽

A Carreón-Rodríguez ◽

...

Keyword(s):

Luciferase Reporter ◽

Transcriptional Level ◽

Mrna Levels ◽

Mobility Shift ◽

Camp Response Element ◽

Glucocorticoid Response Element ◽

Response Element ◽

Nuclear Extracts ◽

Glucocorticoid Response ◽

Hypothalamic Cells

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (− 776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (− 101/− 94 and − 59/− 52) and glucocorticoid response element (GRE) half-site (− 210/− 205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (− 101/− 94) but not to CRE (− 59/− 52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

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Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

Biochemical Journal ◽

10.1042/bcj20180394 ◽

2019 ◽

Vol 476 (8) ◽

pp. 1247-1266

Author(s):

Zhanjun Hou ◽

Carrie O'Connor ◽

Josephine Frühauf ◽

Steve Orr ◽

Seongho Kim ◽

...

Keyword(s):

Reporter Gene ◽

Transcriptional Control ◽

Consensus Sequence ◽

Minimal Promoter ◽

Mobility Shift ◽

Nuclear Extracts ◽

Ht1080 Cells ◽

Promoter Construct ◽

Mobility Shift Assay ◽

Reporter Gene Assays

Abstract Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94. Progressive deletions of the hPCFT promoter construct (−2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions −50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.

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Loading of DNA-Binding Factors to an Erythroid Enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.20.6.1993-2003.2000 ◽

2000 ◽

Vol 20 (6) ◽

pp. 1993-2003 ◽

Cited By ~ 16

Author(s):

Shau-Ching Wen ◽

Karim Roder ◽

Kuang-Yu Hu ◽

Irene Rombel ◽

Narender R. Gavva ◽

...

Keyword(s):

Regulatory Element ◽

K562 Cells ◽

Globin Genes ◽

Erythroid Cells ◽

Binding Studies ◽

Mobility Shift ◽

Specific Expression ◽

Factor Binding ◽

Globin Promoter

ABSTRACT The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human α-like globin genes, the embryonic ζ and the adult α2/α/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3′-NA, modulates the capability of HS-40 to activate the embryonic ζ-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. First, human erythroid K562 cells stably integrated with various HS-40 mutants cis linked to a human α-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome. Second, a specific 1-bp, GC→TA mutation in the 3′-NA motif of HS-40, 3′-NA(II), has been shown previously to cause significant derepression of the embryonic ζ-globin promoter activity in erythroid cells. This derepression was hypothesized to be regulated through competitive binding of different nuclear factors, in particular AP1 and NF-E2, to the 3′-NA motif. By gel mobility shift and transient cotransfection assays, we now show that 3′-NA(II) mutation completely abolishes the binding of small MafK homodimer. Surprisingly, NF-E2 as well as AP1 can still bind to the 3′-NA(II) sequence. The association constants of both NF-E2 and AP1 are similar to their interactions with the wild-type 3′-NA motif. However, the 3′-NA(II) mutation causes an approximately twofold reduction of the binding affinity of NF-E2 factor to the 3′-NA motif. This reduction of affinity could be accounted for by a twofold-higher rate of dissociation of the NF-E2–3′-NA(II) complex. Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human α-globin locus via the 3′-NA motif of HS-40 in embryonic/fetal erythroid cells. Furthermore, extrapolation of the in vitro binding studies suggests that factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.

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Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3002 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3002-3014 ◽

Cited By ~ 74

Author(s):

K Kudrycki ◽

C Stein-Izsak ◽

C Behn ◽

M Grillo ◽

R Akeson ◽

...

Keyword(s):

Nuclear Protein ◽

Consensus Sequence ◽

Binding Motif ◽

Sequence Motif ◽

Olfactory Neuron ◽

Sequence Motifs ◽

Mobility Shift ◽

Specific Expression ◽

Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

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A 5′ Regulatory Sequence Containing Two Ets Motifs Controls the Expression of the Wiskott-Aldrich Syndrome Protein (WASP) Gene in Human Hematopoietic Cells

Blood ◽

10.1182/blood.v91.12.4554.412k26_4554_4560 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4554-4560 ◽

Cited By ~ 1

Author(s):

A. Petrella ◽

I. Doti ◽

V. Agosti ◽

P. Carandente Giarrusso ◽

D. Vitale ◽

...

Keyword(s):

Transcription Factors ◽

Jurkat Cells ◽

Regulatory Sequence ◽

Regulatory Sequences ◽

Specific Expression ◽

Wiskott Aldrich Syndrome ◽

Nuclear Extracts ◽

Ets Family ◽

Wasp Gene ◽

Syndrome Protein

The recently-identified Wiskott-Aldrich syndrome protein gene (WASP) is responsible for the Wiskott-Aldrich X-linked immunodeficiency as well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP coding sequence. Transfection experiments showed that this fragment is capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human hematopoietic cell lines tested. Progressive 5′ deletions showed that the minimal sequence required for hematopoietic-specific expression consists of 137 bp upstream of the transcription start site. This contains potential binding sites for several hematopoietic transcription factors and, in particular, two Ets-1 consensus that proved able to specifically bind to proteins present in nuclear extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in transactivation of the CAT reporter gene under the control of WASP regulatory sequences. Disruption of the Ets-binding sequences by side-directed mutagenesis abolished CAT expression in Jurkat cells, indicating that transcription factors of the Ets family play a key role in the control of WASP transcription.

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3,5-Diiodo-L-thyronine (T2) has selective thyromimetic effects in vivo and in vitro

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0190137 ◽

1997 ◽

Vol 19 (2) ◽

pp. 137-147 ◽

Cited By ~ 60

Author(s):

SG Ball ◽

J Sokolov ◽

WW Chin

Keyword(s):

Gh3 Cells ◽

Mrna Levels ◽

Thyroid Status ◽

Hormone Activity ◽

Mobility Shift ◽

Nuclear Extracts ◽

Electrophoretic Mobility Shift Assays ◽

Gh3 Cell

Recent data have suggested that the iodothyronine, 3,5-diiodo-l-thyronine (T2), has selective thyromimetic activity. In vivo, T2 has been shown to suppress TSH levels at doses that do not produce significant peripheral manifestations of thyroid hormone activity. Furthermore, T2 has been shown to produce smaller increments in peripheral indices of thyroid status than does T3, when doses resulting in equivalent suppression of circulating TSH are compared. We have assessed the selective thyromimetic activity of T2 in vivo and in vitro, and performed in vitro studies to assess the potential molecular basis for these selective properties. T2 was 100-fold less potent than T3 in stimulating GH mRNA levels in GH3 cells. In contrast, the iodothyronines were almost equivalent in their ability to downregulate TRbeta2 mRNA levels in this cell line. Both 3,3'-diiodo-L-thyronine and thyronine exhibited no significant thyromimetic effects on either process. In vivo, doses of T2 and T3 that were equivalent in their induction of hepatic malic enzyme (ME) mRNA did not produce equivalent suppression of circulating TSH, with T2 being only 27% as effective as T3. T2 was up to 500-fold less potent than T3 in displacing [125I]-T3 from in vitro translated specific nuclear receptors (TRs) and GH3 cell nuclear extracts. Electrophoretic mobility shift assays, assessing the ability of T2 to produce dissociation of TRbeta1 homodimers from inverted palindrome T3 response elements, indicated that T2 was also 1000-fold less potent than T3 in this respect. These data confirm that T2 has significant thyromimetic activity, and that this activity is selective both in vivo and in vitro. However, there are no data to support a selective central effect, T2 being relatively more potent in stimulating hepatic ME mRNA than in suppression of TSH in vivo. The basis for this differential thyromimetic activity is not selective affinity of the different TR isoforms for T2, or divergent properties of T2 in competitive binding and functional assays in vitro.

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Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.

Journal of Experimental Medicine ◽

10.1084/jem.178.5.1681 ◽

1993 ◽

Vol 178 (5) ◽

pp. 1681-1692 ◽

Cited By ~ 48

Author(s):

L R Gottschalk ◽

D M Giannola ◽

S G Emerson

Keyword(s):

T Cells ◽

Transcription Factors ◽

T Cell ◽

Protein Binding ◽

Binding Site ◽

Binding Sites ◽

Nuclear Protein ◽

Mobility Shift ◽

Nuclear Extracts ◽

Mobility Shift Assay

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to < 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

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