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Query: UNIPROT:P06889 (Mol)
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We have been studying the molecular mechanism of neuronal differentiation through which the multipotent precursor becomes limited to the final transmitter phenotype. Here we focused on the role of the 5' proximal regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gene in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene. These motifs were sufficient to confer activity-dependent expression of the gene during neurodifferentiation when it was tested using transient transfection assays of primary developing spinal cord neurons treated with tetrodotoxin (TTX). This region was then used as a DNA probe in mobility shift assays, with nuclear proteins derived from phenotypically and ontogenetically distinct brain regions. Only a few low abundance protein-DNA complexes were detected and only with nuclear proteins derived from developing but not from adult brain. The spatiotemporal pattern of these complexes did not show correlation with enkephalin expression which was assessed by RT-PCR. We employed synthetic probes corresponding to consensus as well as ENK-specific sequences of the individual motifs to identify the nature of the observed bands. Although both consensus NF1 and enkCRE1(NF1) formed complexes with nuclear proteins derived from the striatum and cortex at various ages, the appearance of the bands was not correlated with ENK expression. Surprisingly, no complexes were detected if other ENK-specific motifs were used as probes. We also tested nuclear extracts derived from forskolin-induced and control C6 glioma cells, again using the whole proximal regulatory cassette as well as individual motifs. These experiments showed the formation of elaborate protein-DNA bands. There was no direct correlation between the appearance of bands and forskolin-induced ENK expression. Unexpectedly, all ENK-specific motifs formed specific and highly abundant protein-DNA complexes when nuclear extracts from the human tumor cell line (HeLa), which does not express ENK, were used. Based on these observations, we concluded that: 1. Interactions between the proximal regulatory cassette and additional probably far distant regions of the rENK gene and their binding proteins may be necessary to confer developmentally regulated, cell-specific expression of the ENK gene; and 2. Inducibility of the gene by common cis-elements can be governed by this region; however, the cell-specificity of the induction remains elusive.
Mol Neurobiol
PMID:Protein-DNA interactions during phenotypic differentiation. 757 7

In this report we identify novel spliced forms of cyclic AMP (cAMP) response element-binding protein-1 (CREB-1) mRNA. These forms contained an additional 17 nucleotide insert, which we refer to as the beta exon, located between exons 4 and 7 of the delta, and 5 and 7 of the alpha forms of CREB-1 transcript (nomenclature of Ruppert et al. 1992; EMBO Journal 11, 1503-1512). The inclusion of the beta exon led to the generation of mRNAs in which the frame of CREB-1 sequences 3' to the exon was shifted such that the encoded proteins terminate after the transactivation domain, but before the target serine for cAMP-dependent protein kinase. The beta exon-containing CREB-1 mRNAs were more abundant in tissues that respond poorly to cAMP, suggesting that the generation of beta CREB-1 mRNAs may contribute to the down-regulation of CREB-1 activity and cAMP responsiveness.
J Mol Endocrinol 1995 Apr
PMID:A novel cyclic AMP response element-binding protein-1 (CREB-1) splice product may down-regulate CREB-1 activity. 761 8

We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.
Mol Cell Biol 1995 Sep
PMID:A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist. 765 29

Tat, the transactivator protein encoded by HIV-1, acts in vivo to increase transcriptional initiation and stabilize elongation. We examined the effects of purified, bacterially-expressed Tat on HIV-1 transcription in a cell-free system. Tat specifically stimulated HIV-directed transcription 12-fold in HeLa cell nuclear extracts and this effect was principally due to increased transcriptional elongation. The degree of transactivation was greatest at later times during the transcription reaction when basal levels of transcription were reduced. At early times, the proportion of basal transcriptional complexes that elongate efficiently was high. Ongoing transcription increased the number of complexes requiring Tat for efficient elongation, possibly due to the activation of a repressor(s). To examine this hypothesis, the effects of the detergent Sarkosyl on HIV transcription were studied. Sarkosyl stimulated HIV-1 transcription to a level similar to that occurring in the presence of Tat alone by improving elongation. Transcription was elevated by Sarkosyl at concentrations inhibitory to reinitiation indicating that inefficient elongation is due to transcriptional pausing. Transcriptional stimulation by Sarkosyl was a general phenomenon as it was also observed with heterologous eukaryotic promoters. Tat was capable of stimulating elongation from a heterologous promoter when Tat binding was provided by a downstream TAR element. We propose that Tat acts as a general transcription factor whose binding at the promoter overcomes inefficient transcriptional elongation.
J Mol Biol 1993 Aug 05
PMID:HIV-1 Tat overcomes inefficient transcriptional elongation in vitro. 768 12

We have investigated the molecular basis of the variability of the somatostatin cAMP response element (CRE) function in different cell lines. All cells tested contain detectable levels of the CRE-binding protein CREB-1, which mediates transactivation in response to the cAMP-dependent protein kinase (protein kinase-A), in forms that can bind to a somatostatin CRE. Although both responsive and nonresponsive cells contain CREB-1 in heterodimers with activating transcription factor-1 (ATF-1), only cells that allow a cAMP response have a significant proportion of CREB-1 in a homodimeric form. Transfection experiments demonstrate that ATF-1 is capable of antagonizing CREB-1-dependent activation, suggesting that the ability of CREB-1 to mediate a cAMP response is down-regulated by heterodimer formation with ATF-1.
Mol Endocrinol 1995 Feb
PMID:Activating transcription factor-1 is a specific antagonist of the cyclic adenosine 3'.5'-monophosphate (cAMP) response element-binding protein-1-mediated response to cAMP. 777 75

Transcription factor CREB regulates cyclic AMP (cAMP)-dependent gene expression by binding to and activating transcription from cAMP response elements (CREs) in the promoters of target genes. The transcriptional transactivation functions of CREB are activated by its phosphorylation by cAMP-dependent protein kinase A (PKA). In studies of many different phenotypically distinct cells, the CRE of the somatostatin gene promoter is a prototype of a highly cAMP-responsive element regulated by CREB. We now report on a somatostatin-producing rat insulinoma cell line, RIN-1027-B2, in which transcription from the somatostatin gene promoter is paradoxically repressed by CREB. We find that CREB fails to transactivate a CRE-containing somatostatin-chloramphenicol acetyltransferase reporter even when coexpressed with the catalytic subunit of PKA. CAAT box/enhancer-binding protein beta (C/EBP beta) and C/EBP-related activating transcription factor bind to the CRE in the promoter of the somatostatin gene and transactivate transcription. CREB binds competitively with C/EBP beta to the somatostatin CRE in vitro and represses C/EBP beta-induced transcription of the CRE-containing somatostatin-chloramphenicol acetyltransferase reporter. The lack of CREB-mediated transcriptional stimulation is due to the presence of a heat-stable inhibitor of PKA that prevents activation of PKA and subsequent CREB phosphorylation in the nucleus. These findings indicate that dephosphorylated CREB is a negative regulator of C/EBP-activated transcription of the somatostatin gene promoter in RIN-1027-B2 cells.
Mol Cell Biol 1995 Jan
PMID:Impaired cyclic AMP-dependent phosphorylation renders CREB a repressor of C/EBP-induced transcription of the somatostatin gene in an insulinoma cell line. 779 50

In adult rats, the expression of transcription factor proteins c-Jun and CREB and their colocalization with tyrosine hydroxylase (TH) were investigated in neurons of the substantia nigra compacta (SNC) axotomized by stereotaxic unilateral transection of the medial forebrain bundle (MFB). Axotomized SNC neurons were identified by injection of the retrograde tracer horseradish-peroxidase-coupled-gold (HRP-gold) into the ipsilateral striatum 5 days prior to MFB transection. Nuclear c-Jun immunoreactivity (IR) appeared 36 h after MFB transection in SNC neurons, was maximal after 5 days, and declined after 10 days. c-Jun-IR was visible in HRP-gold-labeled SNC neurons, demonstrating that c-Jun is in fact expressed in axotomized neurons. The constitutively expressed CREB (calcium/cAMP response element-binding protein, syn. CREB-1) was present in apparently all neuronal and glial cells in the brains of untreated rats including those SNC neurons that coexpressed TH. Three days following MFB transection, the nuclear CREB-IR disappeared in the axotomized SNC neurons labeled by TH-IR and was almost completely absent after 20 days in this neuronal population. The TH-IR rapidly declined 5 days after MFB transection, and 10 and 100 days post-axotomy the number of TH-labeled neurons was reduced by 52 and 80%, respectively. During this period, the majority of surviving TH positive neurons coexpressed c-Jun but were immunonegative for CREB. Between 3 and 60 days following MFB transection, the number of CREB-labeled glial cell nuclei increased in the ipsilateral substantia nigra by about 80%. Concomitantly, expression of GFAP, a marker protein for astrocytes, was also enhanced whereas nuclear c-Jun-, JunD-, and c-Fos-IR did not change in glial cells. These findings demonstrate that c-Jun can be expressed in axotomized neurons during the absence of CREB and suggest a role of c-Jun in the transcriptional control of the TH gene.
Mol Cell Neurosci 1994 Oct
PMID:Induction of c-Jun and suppression of CREB transcription factor proteins in axotomized neurons of substantia nigra and covariation with tyrosine hydroxylase. 782 Mar 66

Two cis-acting promoter elements (-108 to -100 and -49 to -33) of the mouse alpha A-crystallin gene, which is highly expressed in the ocular lens, were studied. Here we show that DE1 (-108 to -100; 5'TGACGGTG3'), which resembles the consensus cyclic AMP (cAMP)-responsive element sequence (CRE; 5'TGACGT[A/C][A/G]3'), behaves like a functional CRE site. Transfection experiments and electrophoretic mobility shift assays (EMSAs) using site-specific mutations correlated a loss of function with deviations from the CRE consensus sequence. Results of EMSAs in the presence of antisera against CREB, delta CREB, and CREM were consistent with the binding of CREB-like proteins to the DE1 sequence. Stimulation of alpha A-crystallin promoter activity via 8-bromo-cAMP, forskolin, or human T-cell leukemia virus type I Tax1 in transfections and reduction of activity of this site in cell-free transcription tests by competition with the somatostatin CRE supported the idea that DE1 is a functional CRE. Finally, Pax-6, a member of the paired-box family of transcription factors, activated the mouse alpha A-crystallin promoter in cotransfected COP-8 fibroblasts and bound to the -59 to -29 promoter sequence in EMSAs. These data provide evidence for a synergistic role of Pax-6 and CREB-like proteins for high expression of the mouse alpha A-crystallin gene in the lens.
Mol Cell Biol 1995 Feb
PMID:Transcriptional regulation of the mouse alpha A-crystallin gene: activation dependent on a cyclic AMP-responsive element (DE1/CRE) and a Pax-6-binding site. 782 34

The present study has investigated whether nerve fiber transection alters the phosphorylation of serine at position 133 (Ser133) of the transcription factor CREB (phosphoCREB). Activation of CREB by phosphorylation has a major function in the control of gene transcription. PhosphoCREB was visualized by antisera that specifically react with an epitope comprising the phosphorylated Ser133 of CREB as well as of CREM and ATF1 proteins. In untreated rats, nuclear immunoreactivity (IR) of phosphoCREB was consistently visible, e.g. in the cortex, thalamic and hypothalamic compartments and central termination areas of primary somatosensory afferents. Transection of peripheral (sciatic nerve), cranial (hypoglossal and facial nerve) and central (medial forebrain bundle and mammillo-thalamic tract) nerve fibers did not increase phosphoCREB-IR in the axotomized neurons between 5 min and 30 days post-axotomy. In contrast, phosphoCREB-IR appeared after 24 h in glial cells adjacent to the axotomized motoneurons and persisted up to 4 weeks. This increase in glial phosphoCREB-IR was paralleled by enhanced expression of the CREB protein itself. Between 20 min and 24 h following sciatic nerve transection, the number of phosphoCREB labeled nuclei also increased in neurons of the ipsilateral superficial dorsal horn of lumbar L3-L5 spinal cord segments. These data suggest that phosphorylation of Ser133 in CREB/CREM/ATF1 proteins is not involved in the transcriptional control of early-response genes such as c-jun in axotomized neurons following nerve transection. This is in contrast to the reported phosphorylation of CREB and its trans-acting effects on immediate-early genes such as c-fos after transynaptic neuronal excitation.
Brain Res Mol Brain Res 1994 Oct
PMID:The transcription factor CREB is not phosphorylated at serine 133 in axotomized neurons: implications for the expression of AP-1 proteins. 785 55

Cyclic AMP (cAMP) stimulates the expression of numerous genes through the protein kinase A (PK-A)-mediated phosphorylation of the nuclear factor CREB at Ser-133 (G. A. Gonzalez and M. R. Montminy, Cell 59:675-680, 1989). Like other signal transduction pathways, cAMP induces gene expression with burst-attenuation kinetics; cAMP-dependent transcription and CREB phosphorylation peak within 30 min and decline steadily over the next 4 to 6 h via the protein phosphatase 1-mediated dephosphorylation of CREB (M. Hagiwara, A. Alberts, P. Brindle, J. Meinkoth, J. Feramisco, T. Deng, M. Karin, S. Shenolikar, and M. Montminy, Cell 70:105-113, 1992). Here we characterize a third phase in cAMP-responsive transcription--a refractory period during which hormone-treated cells become transcriptionally unresponsive to subsequent stimulation by cAMP. This refractory period begins 6 to 8 h after stimulation and lasts 3 to 5 days after the removal of hormone. In contrast to the earlier attenuation phase, transcription of cAMP-responsive genes during the refractory period is not restored by inhibitors of protein phosphatase 1 activity. Rather, the establishment and maintenance of this phase rely on a marked reduction in PK-A catalytic subunit expression at the translational level. As overexpression of C-subunit protein can reactive transcription of cAMP-responsive genes during the refractory period, our results suggest that hormone-responsive cells may stimulate, attenuate, and then silence signal-dependent genes through distinct regulatory mechanisms.
Mol Cell Biol 1995 Mar
PMID:A refractory phase in cyclic AMP-responsive transcription requires down regulation of protein kinase A. 786 72


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