Cholesterogenic Lanosterol 14α-Demethylase (CYP51) Is an Immediate Early Response Gene

Lanosterol 14α-demethylase (CYP51) responds to cholesterol feedback regulation through sterol regulatory element binding proteins (SREBPs). The proximal promoter of CYP51 contains a conserved region with clustered regulatory elements: GC box, cAMP-response elements (CRE-like), and sterol regulatory element (SRE). In lipid-rich (SREBP-poor) conditions, the CYP51 mRNA drops gradually, the promoter activity is diminished, and no DNA-protein complex is observed at the CYP51-SRE1 site. The majority of cAMP-dependent transactivation is mediated through a single CRE (CYP51-CRE2). Exposure of JEG-3 cells to forskolin, a mediator of the cAMP-dependent signaling pathway, provokes an immediate early response of CYP51, which has not been described before for any cholesterogenic gene. The CYP51 mRNA increases up to 4-fold in 2 h and drops to basal level after 4 h. The inducible cAMP early repressor (ICER) is involved in attenuation of transcription. Overexpressed CRE-binding protein (CREB)/CRE modulator (CREM) transactivates the mouse/human CYP51 promoters containing CYP51-CRE2 independently of SREBPs, and ICER decreases the CREB-induced transcription. Besides the increased CYP51 mRNA, forskolin affects the de novo sterol biosynthesis in JEG-3 cells. An increased consumption of lanosterol, a substrate of CYP51, is observed together with modulation of the postlanosterol cholesterogenesis, indicating that cAMP-dependent stimuli cross-talk with cholesterol feedback regulation. CRE-2 is essential for cAMP-dependent transactivation, whereas SRE seems to be less important. Interestingly, when CREB is not limiting, the increasing amounts of SREBP-1a fail to transactivate the CYP51 promoter above the CREB-only level, suggesting that hormones might have an important role in regulating cholesterogenesis in vivo.

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Intestinal perfusion induces rapid activation of immediate-early genes in weaning rats

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.2001.281.4.r1274 ◽

2001 ◽

Vol 281 (4) ◽

pp. R1274-R1282 ◽

Cited By ~ 8

Author(s):

Lan Jiang ◽

Heather Lawsky ◽

Relicardo M. Coloso ◽

Mary A. Dudley ◽

Ronaldo P. Ferraris

Keyword(s):

Mrna Expression ◽

Target Genes ◽

De Novo ◽

Immediate Early Genes ◽

Regulatory Elements ◽

Intestinal Perfusion ◽

Immediate Early ◽

Mrna Levels ◽

Early Genes

C- fos and c- jun are immediate-early genes (IEGs) that are rapidly expressed after a variety of stimuli. Products of these genes subsequently bind to DNA regulatory elements of target genes to modulate their transcription. In rat small intestine, IEG mRNA expression increases dramatically after refeeding following a 48-h fast. We used an in vivo intestinal perfusion model to test the hypothesis that metabolism of absorbed nutrients stimulates the expression of IEGs. Compared with those of unperfused intestines, IEG mRNA levels increased up to 11 times after intestinal perfusion for 0.3–4 h with Ringer solutions containing high (100 mM) fructose (HF), glucose (HG), or mannitol (HM). Abundance of mRNA returned to preperfusion levels after 8 h. Levels of c- fos and c- jun mRNA and proteins were modest and evenly distributed among enterocytes lining the villi of unperfused intestines. HF and HM perfusion markedly enhanced IEG mRNA expression along the entire villus axis. The perfusion-induced increase in IEG expression was inhibited by actinomycin-D. Luminal perfusion induces transient but dramatic increases in c- fos and c- jun expression in villus enterocytes. Induction does not require metabolizable or absorbable nutrients but may involve de novo gene transcription in cells along the villus.

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Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.206-216.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 206-216

Author(s):

Y T Chung ◽

E B Keller

Keyword(s):

Drosophila Melanogaster ◽

Transient Expression ◽

Promoter Activity ◽

Regulatory Element ◽

Regulatory Elements ◽

Proximal Promoter ◽

Base Pairs ◽

Chloramphenicol Acetyltransferase Gene ◽

Constitutive Synthesis

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.

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Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.206 ◽

1990 ◽

Vol 10 (1) ◽

pp. 206-216 ◽

Cited By ~ 29

Author(s):

Y T Chung ◽

E B Keller

Keyword(s):

Drosophila Melanogaster ◽

Transient Expression ◽

Promoter Activity ◽

Regulatory Element ◽

Regulatory Elements ◽

Proximal Promoter ◽

Base Pairs ◽

Chloramphenicol Acetyltransferase Gene ◽

Constitutive Synthesis

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A Single Pitx1 Binding Site Is Essential for Activity of the LHβ Promoter in Transgenic Mice

Molecular Endocrinology ◽

10.1210/mend.15.5.0628 ◽

2001 ◽

Vol 15 (5) ◽

pp. 734-746 ◽

Cited By ~ 3

Author(s):

Christine C. Quirk ◽

Kristen L. Lozada ◽

Ruth A. Keri ◽

John H. Nilson

Keyword(s):

Transgenic Mice ◽

Binding Site ◽

Cell Lines ◽

Regulatory Element ◽

Regulatory Elements ◽

Vital Role ◽

Proximal Region ◽

Expression Vectors ◽

Homeodomain Protein

Abstract Reproduction depends on regulated expression of the LHβ gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHβ promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHβ gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LβT2), an LHβ promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHβ promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHβ promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.

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Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate-early gene expression via CArG elements.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4243 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4243-4255 ◽

Cited By ~ 119

Author(s):

D Gius ◽

X M Cao ◽

F J Rauscher ◽

D R Cohen ◽

T Curran ◽

...

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Transcriptional Activation ◽

Leucine Zipper ◽

Regulatory Element ◽

Regulatory Elements ◽

Early Gene ◽

Immediate Early ◽

Striking Similarity ◽

Independent Functions

The Fos-Jun complex has been shown to activate transcription through the regulatory element known as the AP-1 binding site. We show that Fos down regulates several immediate-early genes (c-fos, Egr-1, and Egr-2) after mitogenic stimulation. Specifically, we demonstrate that the target for this repression is a sequence of the form CC(A/T)6GG, also known as a CArG box. Whereas Fos bound to the AP-1 site through a domain rich in basic amino acids and associated with Jun via a leucine zipper interaction, mutant Fos proteins lacking these structures were still capable of causing repression. Furthermore, Jun neither enhanced nor inhibited down regulation by Fos. Critical residues required for repression are located within the C-terminal 27 amino acids of c-Fos, since v-Fos and C-terminal truncations of c-Fos did not down regulate. In addition, transfer of 180 c-Fos C-terminal amino acids to Jun conferred upon it the ability to repress. Finally, Fra-1, a Fos-related protein which has striking similarity to Fos in its C-terminal 40 amino acids, also down regulated Egr-1 expression. Thus, Fos is a transcriptional regulator that can activate or repress gene expression by way of two separate functional domains that act on distinct regulatory elements.

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CRISPR/Cas9-mediated editing of Δ5 and Δ6 desaturases impairs Δ8-desaturation and docosahexaenoic acid synthesis in Atlantic salmon (Salmo salar L.)

Scientific Reports ◽

10.1038/s41598-019-53316-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Alex K. Datsomor ◽

Rolf E. Olsen ◽

Nikola Zic ◽

Angelico Madaro ◽

Atle M. Bones ◽

...

Keyword(s):

Atlantic Salmon ◽

Regulatory Element ◽

Acid Synthesis ◽

Fatty Acyl ◽

Chain Polyunsaturated Fatty Acid ◽

Pufa Diet ◽

Element Binding Protein ◽

Sterol Regulatory Element ◽

High Degree

AbstractThe in vivo functions of Atlantic salmon fatty acyl desaturases (fads2), Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2 in long chain polyunsaturated fatty acid (LC-PUFA) synthesis in salmon and fish in general remains to be elucidated. Here, we investigate in vivo functions and in vivo functional redundancy of salmon fads2 using two CRISPR-mediated partial knockout salmon, Δ6abc/5Mt with mutations in Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2, and Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c. F0 fish displaying high degree of gene editing (50–100%) were fed low LC-PUFA and high LC-PUFA diets, the former containing reduced levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids but higher content of linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids, and the latter containing high levels of 20:5n-3 and 22:6n-3 but reduced compositions of 18:2n-6 and 18:3n-3. The Δ6abc/5Mt showed reduced 22:6n-3 levels and accumulated Δ6-desaturation substrates (18:2n-6, 18:3n-3) and Δ5-desaturation substrate (20:4n-3), demonstrating impaired 22:6n-3 synthesis compared to wildtypes (WT). Δ6bcMt showed no effect on Δ6-desaturation compared to WT, suggesting Δ6 Fads2-a as having the predominant Δ6-desaturation activity in salmon, at least in the tissues analyzed. Both Δ6abc/5Mt and Δ6bcMt demonstrated significant accumulation of Δ8-desaturation substrates (20:2n-6, 20:3n-3) when fed low LC-PUFA diet. Additionally, Δ6abc/5Mt demonstrated significant upregulation of the lipogenic transcription regulator, sterol regulatory element binding protein-1 (srebp-1) in liver and pyloric caeca under reduced dietary LC-PUFA. Our data suggest a combined effect of endogenous LC-PUFA synthesis and dietary LC-PUFA levels on srebp-1 expression which ultimately affects LC-PUFA synthesis in salmon. Our data also suggest Δ8-desaturation activities for salmon Δ6 Fads2 enzymes.

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SREBP1 drives KRT80-dependent cytoskeletal changes and invasive behavior in endocrine resistant ERα breast cancer

10.1101/380634 ◽

2018 ◽

Cited By ~ 2

Author(s):

Ylenia Perone ◽

Aaron J. Farrugia ◽

Alba Rodriguez Meira ◽

Balázs Győrffy ◽

Charlotte Ion ◽

...

Keyword(s):

Breast Cancer ◽

Cell Invasion ◽

De Novo ◽

Regulatory Element ◽

Leading Edge ◽

Clinical Endpoints ◽

Breast Cancer Relapse ◽

Element Binding Protein ◽

Cytoskeletal Rearrangements

AbstractApproximately 30% of women diagnosed with ERα breast cancer relapse with metastatic disease following adjuvant treatment with endocrine therapies1,2. The connection between acquisition of drug resistance and invasive potential is poorly understood. In this study, we demonstrate that the type II keratin topological associating domain (TAD)3 undergoes epigenetic reprogramming in cells that develop resistance to aromatase inhibitors (AI), leading to keratin 80 (KRT80) upregulation. In agreement, an increased number of KRT80-positive cells are observed at relapse in vivo while KRT80 expression associates with poor outcome using several clinical endpoints. KRT80 expression is driven by de novo enhancer activation by sterol regulatory element-binding protein 14 (SREBP1). KRT80 upregulation directly promotes cytoskeletal rearrangements at the leading edge, increased focal adhesion maturation and cellular stiffening, which collectively promote cancer cell invasion. Shear-wave elasticity imaging of prospective patients shows that KRT80 levels correlate with stiffer tumors in vivo. Collectively, our data uncover an unpredicted and potentially targetable direct link between epigenetic and cytoskeletal reprogramming promoting cell invasion in response to chronic AI treatment.

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Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

Bioscience Reports ◽

10.1042/bsr20120095 ◽

2012 ◽

Vol 33 (1) ◽

Cited By ~ 6

Author(s):

Lidia A. Daimiel ◽

María E. Fernández-Suárez ◽

Sara Rodríguez-Acebes ◽

Lorena Crespo ◽

Miguel A. Lasunción ◽

...

Keyword(s):

Cellular Response ◽

Binding Protein ◽

Regulatory Element ◽

Cholesterol Biosynthesis ◽

Regulatory Elements ◽

Proximal Promoter ◽

Luciferase Reporter ◽

Mobility Shift ◽

Gene Characterization ◽

Cis Acting

DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.

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Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽

10.1242/dev.125.22.4349 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4349-4358 ◽

Cited By ~ 4

Author(s):

J. Charite ◽

W. de Graaff ◽

D. Consten ◽

M.J. Reijnen ◽

J. Korving ◽

...

Keyword(s):

Binding Sites ◽

Hox Genes ◽

Regulatory Element ◽

Ectopic Expression ◽

Positional Information ◽

Regulatory Elements ◽

Gene Products ◽

Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

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