Differentiation-responsive elements in the 5' region of the mouse tissue plasminogen activator gene confer two-stage regulation by retinoic acid and cyclic AMP in teratocarcinoma cells

F9 cells induced to differentiate with retinoic acid (RA) increase transcription of the tissue plasminogen activator (t-PA) gene. Further treatment of these cells with cyclic AMP (cAMP) results in an additional stimulation of t-PA gene transcription. To investigate the mechanism of this two-stage regulation, 4 kilobase pairs (kbp) of 5'-flanking sequence from the murine t-PA gene was isolated. Two major start sites for transcription were found, neither of which depended on a classical TATA motif for correct initiation. By using transient transfection assays, it was determined that 4-kbp of flanking sequence could confer on reporter genes the same two-stage differentiation-specific expression as was observed for the endogenous t-PA gene. Deletion analyses of this 4-kbp fragment showed that 190 bp of flanking sequence was sufficient to bestow the same degree of two-stage regulation on reporter gene constructs. Within this region of DNA, sequence analysis revealed a possible cAMP regulatory element, a CTF/NF-1 recognition sequence, two potential Sp1 sites, and five potential binding sites for transcription factor AP-2. The deletion experiments, coupled with the positions of these potential cis-acting elements, suggest that multiple transcription factors, including those that bind to cAMP regulatory element, CTF/NF-1, Sp1, and AP-2 sites, may be involved in regulation of the t-PA gene during F9 cell differentiation.

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Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5883-5893.1990 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5883-5893

Author(s):

A L Darrow ◽

R J Rickles ◽

L T Pecorino ◽

S Strickland

Keyword(s):

Transcription Factor ◽

Retinoic Acid ◽

Cyclic Amp ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Differentiated Cells ◽

Cell Extracts ◽

Transcription Factor Sp1 ◽

Tissue Plasminogen

The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.

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Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5883 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5883-5893 ◽

Cited By ~ 38

Author(s):

A L Darrow ◽

R J Rickles ◽

L T Pecorino ◽

S Strickland

Keyword(s):

Transcription Factor ◽

Retinoic Acid ◽

Cyclic Amp ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Differentiated Cells ◽

Cell Extracts ◽

Transcription Factor Sp1 ◽

Tissue Plasminogen

The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.

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The mouse tissue plasminogen activator gene 5′ flanking region directs appropriate expression in development and a seizure-enhanced response in the CNS

Development ◽

10.1242/dev.120.11.3173 ◽

1994 ◽

Vol 120 (11) ◽

pp. 3173-3183 ◽

Cited By ~ 4

Author(s):

P.M. Carroll ◽

S.E. Tsirka ◽

W.G. Richards ◽

M.A. Frohman ◽

S. Strickland

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Cranial Nerves ◽

In Situ Analysis ◽

Mouse Tissue ◽

Galactosidase Activity ◽

Tissue Plasminogen ◽

Flanking Sequences ◽

Beta Galactosidase

Tissue plasminogen activator (t-PA) is a secreted serine protease implicated in multiple aspects of development. In the adult rat brain, transcription of t-PA is an immediate-early response in the hippocampus following treatments that induce neuronal plasticity. To study the sequence elements that govern transcription of this gene, in situ analysis was used to define t-PA's temporal and spatial expression pattern in midgestation embryos. Transgenic mice were then generated carrying t-PA 5′ flanking sequences linked to the E. coli lacZ gene. Constructs containing 4 kb of the flanking sequences (4.0TAMGAL) confer beta-galactosidase activity mostly to the same tissues that exhibit high levels of t-PA mRNA by in situ analysis. In 4.0TAMGAL embryos from embryonic day 8.5 (E8.5) to 13.5 (E13.5), the majority of expression observed is localized to neural ectoderm-derived tissues. beta-galactosidase activity is first detected in restricted neuromeres in the midbrain and diencephalon, at E8.5 and E9.5 respectively. At E10.5, transgene expression is observed in neural crest-derived cranial nerves and dorsal root ganglia, but not placode-derived cranial nerves. From E10.5 to E13.5, beta-galactosidase activity is observed in postmitotic neurons of the midbrain, spinal cord, neural retina and the developing olfactory system. beta-galactosidase activity is also detected in areas undergoing tissue remodeling such as the pinna of the ear, whisker follicles and the limbs. In adult mice, lacZ is expressed in the hippocampus and this expression was found to be enhanced upon seizure in the giant pyramidal neurons of CA3. These results reinforce the concept that t-PA plays a role in neurogenesis and morphogenesis, and identifies the promoter region that directs its transcriptional regulation both in development and in the CNS.

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