scholarly journals A silencer inhibitor confers specific expression of intestinal trefoil factor in gobletlike cell lines

2001 ◽  
Vol 280 (6) ◽  
pp. G1114-G1123 ◽  
Author(s):  
Dai Iwakiri ◽  
Daniel K. Podolsky
Keyword(s):  
Cell Lines ◽  
Goblet Cell ◽  
Regulatory Element ◽  
Goblet Cells ◽  
Transient Transfection ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor ◽  
Specific Expression ◽  
Silencer Element

Intestinal trefoil factor (ITF) is selectively expressed in intestinal goblet cells. Previous studies identified cis-regulatory elements in the proximal promoter of ITF, but these were insufficient to recapitulate the exquisite tissue- and cell-specific expression of native ITF in vivo. Preliminary studies suggested that goblet cell-specific expression of murine ITF requires elements far upstream that include a silencer element that effectively prevents ITF expression in non-goblet cells. Transient transfection studies using native or mutant ITF 5′-flanking sequences identified a region that restores expression in goblet cells. This element, designated goblet cell silencer inhibitor (GCSI) element, enables human and murine goblet cell-like cell lines to override the silencing effect of more proximal elements. The GCSI has no intrinsic enhancer activity and regulates expression only when the silencer element is present. Ligation of GCSI and silencer elements to sucrase-isomaltase conferred goblet cell-specific expression. Goblet cells but not non-goblet cells possess a nuclear protein that binds to the GCSI regulatory element (GCSI binding protein; GCSI-BP). Both transient transfection and gel mobility shift assay studies localize the GCSI and GCSI-BP to −2216 to −2204. We conclude that goblet cell-specific transcription of ITF in vivo depends on a regulatory element designated GCSI.

scholarly journals Goblet-cell-specific transcription of mouse intestinal trefoil factor gene results from collaboration of complex series of positive and negative regulatory elements

1999 ◽  
Vol 341 (2) ◽  
pp. 461-472 ◽  
Author(s):  
Hiroshi ITOH ◽  
Nagamu INOUE ◽  
Daniel K. PODOLSKY
Keyword(s):  
Cell Lines ◽  
Goblet Cell ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Goblet Cells ◽  
Regulatory Elements ◽  
Intestinal Cell ◽  
Gene Promoters ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor

Intestinal trefoil factor (ITF) is expressed selectively in intestinal goblet cells. Previous studies of the rat ITF gene identified one cis-regulatory element, designated the goblet-cell-response element (GCRE), present in the proximal region of the promoter. To identify additional cis-regulatory elements responsible for goblet-cell-specific expression, a DNA fragment containing 6353 bp of the 5′-flanking region of the mouse ITF gene was cloned and its promoter activity was examined extensively. In human and murine intestinal-derived cell lines (LS174T and CMT-93), the luciferase activities of a 6.3-kb construct were 5- and 2-fold greater than the smaller 1.8-kb construct, respectively. In contrast, the activity in non-intestinal cell lines (HepG2 and HeLa) was 2-4-fold lower than the smaller construct. In the region downstream from the 1.8-kb position, strong luciferase activities in LS174T and HepG2 cells were observed using a 201-bp construct. Interestingly, increased activity was almost completely suppressed in cells transfected with a 391-bp construct. Detailed analyses of this region revealed the existence of a 11-bp positive regulatory element (-181 to -170; ACCTCTTCCTG) and a 9-bp negative regulatory element (-208 to -200; ATTGACAGA) in addition to the GCRE. All three elements were well conserved among human, rat and mouse ITF gene promoters. In addition, a mutant 1.8-kb construct in which the negative regulatory region was deleted yielded the same approximate luciferase activity as a 6.3-kb construct, suggesting binding of a goblet-cell-specific silencer inhibitor (SI) between -6.3 and -1.8 kb. The SI present in goblet cells may block the silencers' binding to the pre-initiation complex and allow increased transcriptional activity driven by specific and non-specific enhancers. High-level expression of the mouse ITF gene specifically in intestinal goblet cells may be achieved through the combined effects of these regulatory elements.

scholarly journals Molecular cloning of mouse intestinal trefoil factor and its expression during goblet cell changes

1995 ◽  
Vol 311 (1) ◽  
pp. 293-297 ◽  
Author(s):  
M Tomita ◽  
H Itoh ◽  
N Ishikawa ◽  
A Higa ◽  
H Ide ◽  
...  
Keyword(s):  
Goblet Cell ◽  
Goblet Cells ◽  
Recovery Phase ◽  
Nippostrongylus Brasiliensis ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor ◽  
Cell Hyperplasia ◽  
Reverse Transcription Pcr ◽  
Rapid Amplification ◽  
Mitf Expression

A cDNA encoding mouse intestinal trefoil factor (mITF) was successfully cloned and sequenced from the small intestine of C57BL/6 mouse by using the combination of reverse transcription-PCR and rapid amplification of cDNA ends methods. The gene was, similar to rat and human ITFs, mainly expressed in the small and large intestine. The mITF expression was up-regulated during the recovery phase after depletion of goblet cells in acetic acid-induced colitis. On the other hand, the expression in the jejunum was not altered, while goblet cell hyperplasia was induced by Nippostrongylus brasiliensis infection. These results suggest that the mITF expression did not simply correlate with the number of goblet cells. The mITF may play an important role in the maintenance and repair of mucosal function of the rectum. Additionally, the mITF in the jejunum may play a role in alteration of the physicochemical nature of goblet cell mucins, thereby affecting the establishment of intestinal helminths.

scholarly journals Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion.

1993 ◽  
Vol 268 (9) ◽  
pp. 6694-6702
Author(s):  
D.K. Podolsky ◽  
K. Lynch-Devaney ◽  
J.L. Stow ◽  
P. Oates ◽  
B. Murgue ◽  
...  
Keyword(s):  
Goblet Cell ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor ◽  
Specific Expression

scholarly journals Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion

1993 ◽  
Vol 268 (16) ◽  
pp. 12230
Author(s):  
D.K. Podolsky ◽  
K. Lynch-Devaney ◽  
J.L. Stow ◽  
P. Oates ◽  
B. Murgue ◽  
...  
Keyword(s):  
Goblet Cell ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor ◽  
Specific Expression

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

2001 ◽  
Vol 15 (5) ◽  
pp. 734-746 ◽  
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.

scholarly journals Loading of DNA-Binding Factors to an Erythroid Enhancer

2000 ◽  
Vol 20 (6) ◽  
pp. 1993-2003 ◽  
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.

scholarly journals Hypoxia-Inducible Factor 1–Dependent Induction of Intestinal Trefoil Factor Protects Barrier Function during Hypoxia

2001 ◽  
Vol 193 (9) ◽  
pp. 1027-1034 ◽  
Author(s):  
Glenn T. Furuta ◽  
Jerrold R. Turner ◽  
Cormac T. Taylor ◽  
Robert M. Hershberg ◽  
Katrina Comerford ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Barrier Function ◽  
Vascular Endothelial Cells ◽  
Hypoxia Inducible Factor ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor ◽  
Compensatory Mechanisms ◽  
Mobility Shift

Mucosal organs such as the intestine are supported by a rich and complex underlying vasculature. For this reason, the intestine, and particularly barrier-protective epithelial cells, are susceptible to damage related to diminished blood flow and concomitant tissue hypoxia. We sought to identify compensatory mechanisms that protect epithelial barrier during episodes of intestinal hypoxia. Initial studies examining T84 colonic epithelial cells revealed that barrier function is uniquely resistant to changes elicited by hypoxia. A search for intestinal-specific, barrier-protective factors revealed that the human intestinal trefoil factor (ITF) gene promoter bears a previously unappreciated binding site for hypoxia-inducible factor (HIF)-1. Hypoxia resulted in parallel induction of ITF mRNA and protein. Electrophoretic mobility shift assay analysis using ITF-specific, HIF-1 consensus motifs resulted in a hypoxia-inducible DNA binding activity, and loading cells with antisense oligonucleotides directed against the α chain of HIF-1 resulted in a loss of ITF hypoxia inducibility. Moreover, addition of anti-ITF antibody resulted in a loss of barrier function in epithelial cells exposed to hypoxia, and the addition of recombinant human ITF to vascular endothelial cells partially protected endothelial cells from hypoxia-elicited barrier disruption. Extensions of these studies in vivo revealed prominent hypoxia-elicited increases in intestinal permeability in ITF null mice. HIF-1–dependent induction of ITF may provide an adaptive link for maintenance of barrier function during hypoxia.

Intestinal trefoil factor stimulates mucosal repair processes in vitro and in vivo

1995 ◽  
Vol 108 (4) ◽  
pp. A70
Author(s):  
R. Chinery ◽  
R. Playford ◽  
R. Poulsom ◽  
H.M. Cox
Keyword(s):  
Trefoil Factor ◽  
Intestinal Trefoil Factor ◽  
Mucosal Repair ◽  
Repair Processes
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