scholarly journals CDP Binding to Multiple Sites in the Mouse Mammary Tumor Virus Long Terminal Repeat Suppresses Basal and Glucocorticoid-Induced Transcription

2002 ◽  
Vol 76 (5) ◽  
pp. 2168-2179 ◽  
Author(s):  
Quan Zhu ◽  
Jaquelin P. Dudley
Keyword(s):  
Mammary Gland ◽  
Reporter Gene ◽  
Binding Sites ◽  
Mouse Mammary Tumor Virus ◽  
Mammary Gland Development ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Ltr ◽  
Gland Development

ABSTRACT Mouse mammary tumor virus (MMTV) is transcribed at high levels in the lactating mammary gland to ensure transmission of virus from the milk of infected female mice to susceptible offspring. We previously have shown that the transcription factor CCAAT displacement protein (CDP) is expressed in high amounts in virgin mammary gland, yet DNA-binding activity for the MMTV long terminal repeat (LTR) disappears as mammary tissue differentiates during lactation. CDP is a repressor of MMTV expression and, therefore, MMTV expression is suppressed during early mammary gland development. In this study, we have shown using DNase I footprinting and electrophoretic mobility shift assays that there are at least five CDP-binding sites in the MMTV LTR upstream of those previously described in the promoter-proximal negative regulatory element (NRE). Single mutations in two of these upstream sites (+691 or +692 and +735 relative to the first base of the LTR) reduced CDP binding to the cognate sites and elevated reporter gene expression from the full-length MMTV LTR. Combination of a mutation in the promoter-distal NRE with a mutation in the proximal NRE gave approximately additive increases in LTR-reporter gene activity, suggesting that these binding sites act independently. Mutations in several different CDP-binding sites allowed elevation of reporter gene activity from the MMTV promoter in the absence and presence of glucocorticoids, hormones that contribute to high levels of MMTV transcription during lactation by activation of hormone receptor binding to the LTR. In addition, overexpression of CDP in transient-transfection assays suppressed both basal and glucocorticoid-induced LTR-mediated transcription in a dose-dependent manner. These data suggest that multiple CDP-binding sites contribute independently to regulate binding of positive factors, including glucocorticoid receptor, to the MMTV LTR during mammary gland development.

scholarly journals CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland

2000 ◽  
Vol 74 (14) ◽  
pp. 6348-6357 ◽  
Author(s):  
Quan Zhu ◽  
Keqin Gregg ◽  
Mary Lozano ◽  
Jinqi Liu ◽  
Jaquelin P. Dudley
Keyword(s):  
Mammary Gland ◽  
Reporter Gene ◽  
Mammary Tumor ◽  
Mouse Mammary Tumor Virus ◽  
Regulatory Element ◽  
Luciferase Reporter ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Ltr

ABSTRACT Mouse mammary tumor virus (MMTV) transcription is highest in the lactating mammary gland but is detectable in a variety of other tissues. Previous results have shown that MMTV expression is suppressed in lymphoid and other tissues through the binding of the homeodomain-containing repressor special AT-rich binding protein 1 to a negative regulatory element (NRE) in the MMTV long terminal repeat (LTR). Another homeoprotein repressor, CCAAT displacement protein (CDP), also binds to the MMTV NRE, but a role for CDP in MMTV transcriptional suppression has not yet been demonstrated. In this paper, we show that the level of CDP decreases during development of the mammary gland and that this decline in CDP level correlates with the known increase in MMTV expression observed during mammary gland differentiation. Moreover, CDP overexpression was able to suppress MMTV LTR-reporter gene activity up to 20-fold in transient-transfection assays of mouse mammary cells. To determine if this effect was due to direct binding of CDP to the promoter-proximal NRE, we performed DNase I protection assays to map two CDP-binding sites from +835 to +845 and +920 to +931 relative to the first base of the LTR. Mutations engineered into each of these sites decreased CDP binding to the proximal NRE, whereas a combination of these mutations further reduced binding. Subsequently, each of these mutations was introduced into the full-length MMTV LTR upstream of the luciferase reporter gene. Analysis of stable transfectants of LTR constructs showed that CDP binding site mutations in the proximal NRE elevated reporter gene expression two- to sixfold compared to wild-type LTR constructs. Thus, MMTV expression increases during mammary gland development, in part due to decreased CDP levels and CDP binding to the LTR. Together, these experiments provide the first evidence that CDP acts as a repressor of MMTV transcription in the mammary gland.

scholarly journals Mammary Gland Involution Is Delayed by Activated Akt in Transgenic Mice

2001 ◽  
Vol 15 (6) ◽  
pp. 867-881 ◽  
Author(s):  
Kathryn L. Schwertfeger ◽  
Monica M. Richert ◽  
Steven M. Anderson
Keyword(s):  
Mammary Gland ◽  
Transgenic Mice ◽  
Mammary Gland Development ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Mammary Tumor Virus ◽  
Mammary Gland Involution ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Protein Kinase Akt ◽  
Gland Development

Abstract Activation of the antiapoptotic protein kinase Akt is induced by a number of growth factors that regulate mammary gland development. Akt is expressed during mammary gland development, and expression decreases at the onset of involution. To address Akt actions in mammary gland development, transgenic mice were generated expressing constitutively active Akt in the mammary gland under the control of the mouse mammary tumor virus (MMTV) promoter. Analysis of mammary glands from these mice reveals a delay in both involution and the onset of apoptosis. Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of matrix metalloproteinases (MMPs), is prolonged and increased in the transgenic mice, suggesting that disruption of the MMP:TIMP ratio may contribute to the delayed mammary gland involution observed in the transgenic mice.

scholarly journals A 5′ distal palindrome within the mouse mammary tumor virus-long terminal repeat recruits a mammary gland-specific complex and is required for a synergistic response to progesterone plus prolactin

2008 ◽  
Vol 41 (2) ◽  
pp. 75-90 ◽  
Author(s):  
Joseph E Morabito ◽  
Josephine F Trott ◽  
Dorian M Korz ◽  
Heather E Fairfield ◽  
Sarah H Buck ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mammary Gland ◽  
Long Terminal Repeat ◽  
Mammary Tumor ◽  
Mouse Mammary Tumor Virus ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Specific Complex ◽  
Tumor Virus ◽  
Mmtv Ltr

Progesterone (P) and prolactin (PRL) fulfill crucial roles during growth and differentiation of the mammary epithelium, and each has been implicated in the pathogenesis of mammary cancer. We previously identified that these hormones synergistically stimulate the proliferation of mouse mammary epithelial cells in vivo, although the mechanism(s) underlying their cooperative effect are unknown. We now report a novel pathway by which P and PRL synergize to activate transcription from the long terminal repeat (LTR) of the mouse mammary tumor virus-LTR (MMTV-LTR) in T47D breast cancer cells. Using serial 5′ and 3′ deletions of the MMTV-LTR, in addition to selective mutations, we identified that a previously uncharacterized inverted palindrome on the distal enhancer (−941/−930), in addition to a signal transducer and activator of transcription 5 site, was essential for the synergistic activation of transcription by P and PRL. Notably, hormone synergy occurred via a mechanism that was independent of the P receptor DNA-binding elements found in the proximal MMTV-LTR hormone-response element. The palindrome specifically recruited a protein complex (herein termed mammary gland-specific complex) that was almost exclusive to normal and cancerous mammary cells. The synergy between P and PRL occurred via a Janus kinase 2 and c-Src/Fyn-dependent signaling cascade downstream of P and PRL receptors. Combined, our data outline a novel pathway in T47D cells that may facilitate the action(s) of P and PRL during mammary development and breast cancer.

An entire functional mammary gland may comprise the progeny from a single cell

Development ◽  
1998 ◽  
Vol 125 (10) ◽  
pp. 1921-1930 ◽  
Author(s):  
E.C. Kordon ◽  
G.H. Smith
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mammary Gland ◽  
Mammary Tumor ◽  
Mouse Mammary Tumor Virus ◽  
Mammary Epithelial ◽  
Self Renewal ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore, one stem cell might easily account for the renewal of mammary epithelium over several transplant generations.

scholarly journals Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice.

1990 ◽  
Vol 10 (11) ◽  
pp. 5822-5829 ◽  
Author(s):  
S R Ross ◽  
C L Hsu ◽  
Y Choi ◽  
E Mok ◽  
J P Dudley
Keyword(s):  
Transgenic Mice ◽  
Mouse Mammary Tumor Virus ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Ltr ◽  
Inappropriate Expression

Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene.

scholarly journals Mammary Gland Expression of Mouse Mammary Tumor Virus Is Regulated by a Novel Element in the Long Terminal Repeat

1999 ◽  
Vol 73 (1) ◽  
pp. 368-376 ◽  
Author(s):  
Wei Qin ◽  
Tatyana V. Golovkina ◽  
Tao Peng ◽  
Irene Nepomnaschy ◽  
Valeria Buggiano ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Tumor ◽  
Lymphoid Cell ◽  
Lymphoid Tissue ◽  
Mouse Mammary Tumor Virus ◽  
Specific Expression ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Specific Virus

ABSTRACT Mouse mammary tumor virus (MMTV) infects both lymphoid tissue and lactating mammary gland during its infectious cycle, but some endogenous MMTVs are transcribed only in lymphoid cells. We found a lymphoid cell-specific endogenous MMTV that was converted to a milk-borne, infectious virus through recombination with an exogenously transmitted MMTV. The changed expression pattern correlated with the alteration of a single base pair in the long terminal repeat of the lymphoid cell-specific virus. Transgenic mice with the element from either the milk-borne or lymphoid cell-specific virus upstream of the chloramphenicol acetyltransferase reporter gene showed the same pattern of expression as the virus from which the regulatory sequences were derived. Electrophoretic mobility shift assays with mammary cell extracts showed that the site from the milk-borne virus was preferentially bound by a prolactin-inducible factor that poorly bound the altered site from the lymphoid cell-specific virus. The complex that formed on the milk-borne virus-specific oligonucleotide supershifted with anti-Stat5b antibody. Mice lacking either Stat5a or Stat5b had dramatically reduced levels of MMTV transcripts in mammary gland but not in lymphoid tissue. Thus, a member of the STAT family of transcription factors is involved in the tissue-specific expression of mouse mammary tumor virus in vivo. This is the first example of the involvement of a member of the STAT family of transcription factors in the control of tissue-specific expression.

scholarly journals Mouse Mammary Tumor Virus Sequences Responsible for Activating Cellular Oncogenes

1998 ◽  
Vol 72 (12) ◽  
pp. 9428-9435 ◽  
Author(s):  
Sandra L. Grimm ◽  
Steven K. Nordeen
Keyword(s):  
Mammary Tumor ◽  
Mouse Mammary Tumor Virus ◽  
Specific Activity ◽  
Hormonal Stimulation ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Promoter ◽  
Mmtv Ltr ◽  
Inappropriate Expression

ABSTRACT Integration of mouse mammary tumor virus (MMTV) near theint genes results in the inappropriate expression of these proto-oncogenes and initiates events that lead to the formation of mammary adenocarcinomas. In most cases, the MMTV provirus integrates in a transcriptional orientation opposite that of the intgenes. We have used a novel, vector-based system designed to recapitulate the integration of MMTV upstream of the int-2promoter. Compared to a cellular promoter or another retroviral promoter, the MMTV long terminal repeat (LTR) in this configuration is particularly efficacious at activating the int-2 promoter. The sequences responsible for enhancing the activity of theint-2 promoter map to two domains in the 5′ end of the MMTV LTR. One domain is a previously defined element; the second is an element delineated by these studies that acts synergistically with the first. Both of these elements display mammary cell-specific activity. Thus, even though the MMTV promoter itself is weak without hormonal stimulation, viral integration can position the 5′ LTR elements to efficiently activate transcription from cellular proto-oncogenes. Other functional elements in the LTR have little effect on the activation of the int-2 promoter. Even stimulation of the MMTV promoter with steroid hormones only modestly activates transcription from theint-2 promoter, suggesting that the 5′ elements of the LTR are the predominant determinants of the tissue- and orientation-specific activation of cellular promoters by MMTV.

Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice

1990 ◽  
Vol 10 (11) ◽  
pp. 5822-5829
Author(s):  
S R Ross ◽  
C L Hsu ◽  
Y Choi ◽  
E Mok ◽  
J P Dudley
Keyword(s):  
Transgenic Mice ◽  
Mouse Mammary Tumor Virus ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Ltr ◽  
Inappropriate Expression

Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene.

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