Modular structure of cAMP response element binding protein 2 (CREB2).

The transcription factor cAMP-response element binding protein 2 (CREB2), a member of the family of basic region leucine zipper proteins, has been suggested to function in the brain as a repressor of long-term memory. Using recombinant proteins we show that CREB2 binds in vitro to the palindromic cAMP response element derived from the secretogranin II gene. Recent studies of the chromogranin B, secretogranin II and enkephalin genes showed that CREB2 functioned as a repressor of cAMP-induced transcription. We analyzed the ability of CREB2 to repress transcription using model promoters. A molecular dissection of the CREB2 molecule revealed that CREB2 lacks a transferable repressor domain suggesting that CREB2 may function solely as a "passive" transcriptional repressor. In contrast, "active" repressor domains derived from the thyroid hormone receptor alpha or the NK10 zinc finger protein containing a "Krüppel associated box" could be transfered to a heterologous DNA-binding domain and functioned as fusion proteins in repressing transcription of a reporter gene. In addition, a strong activation domain located at the N-terminus was identified in the CREB2 protein suggesting that CREB2 may act as an activator of transcription by binding to different genetic regulatory elements.

Nuclear targeting of cAMP response element binding protein 2 (CREB2).

The transcription factor cAMP response element binding protein 2 (CREB2) belongs to a family of proteins containing a basic region as DNA-binding domain and a leucine zipper as a dimerization domain in its C-terminus. Using indirect immunofluorescence labeling of cells we show that CREB2 is a nuclear protein. To identify the signal(s) required for nuclear targeting of CREB2, various domains of the protein were expressed in COS cells as fusion proteins with glutathione S-transferase and their cellular location assayed by indirect immunofluorescence using antibodies directed against the glutathione S-transferase moiety of the fusion proteins. The results show that the nuclear targeting signal is located in the C-terminal part of the molecule. Deletion mutagenesis revealed that the basic region of CREB2, encompassing amino acids 280 to 300, is sufficient for sorting CREB2 to the nucleus. Single point mutations of basic amino acids within the basic region of CREB2 identified the sequence KKLKK (amino acids 280 to 284) as important for nuclear targeting. Thus, the basic region of CREB2 is necessary not only for tethering CREB2 to DNA but also for sorting CREB2 to the nucleus. However, sequences outside the basic region are additionally required for efficient nuclear sorting of CREB2.

Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure.

The plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo- material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the DeltapH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo- plastids contain functional ribosomes. The detection of the plastid-encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.

Chlorophyll a formation in the chlorophyll b reductase reaction requires reduced ferredoxin.

The reduction of chlorophyllide b and its analogue zinc pheophorbide b in etioplasts of barley (Hordeum vulgare L.) was investigated in detail. In intact etioplasts, the reduction proceeds to chlorophyllide a and zinc pheophorbide a or, if incubated together with phytyldiphosphate, to chlorophyll a and zinc pheophytin a, respectively. In lysed etioplasts supplied with NADPH, the reduction stops at the intermediate step of 7(1)-OH-chlorophyll(ide) and Zn-7(1)-OH-pheophorbide or Zn-7(1)-OH-pheophytin. However, the final reduction is achieved when reduced ferredoxin is added to the lysed etioplasts, suggesting that ferredoxin is the natural cofactor for reduction of chlorophyll b to chlorophyll a. The reduction to chlorophyll a requires ATP in intact etioplasts but not in lysed etioplasts when reduced ferredoxin is supplied. The role of ATP and the significance of two cofactors for the two steps of reduction are discussed.

Substrate specificity of chlorophyll(ide) b reductase in etioplasts of barley (Hordeum vulgare L.).

Enzyme activity of chlorophyll(ide) b reductase is present in etioplasts. Recently the conversion of chlorophyllide b to chlorophyll a via 7(1)-hydroxychlorophyll a was demonstrated in barley etioplasts. We used zinc pheophorbide b for a detailed investigation of the reduction of the 7-formyl group to the 7(1)-hydroxy compound in intact barley etioplasts. The reaction proceeded likewise before esterification and after esterification with phytyl diphosphate. The metal-free pheophorbide b, that is not accepted by chlorophyll synthase for esterification, is reduced to 7(1)-hydroxypheophorbide a to a small extent. The zinc (13(2)S)-pheophorbide b is at least equally well accepted for reduction as the epimer with the 13(2)R configuration of natural chlorophyll b. The reaction requires NADPH or NADH, although the latter is less effective. ATP is not required for the first step to the 7(1)-hydroxy compound. The significance of chlorophyll b reduction for acclimation from shade to sun leaves and for chlorophyll degradation is discussed.

A (G+C)-rich motif in the aldolase C promoter functions as a constitutive transcriptional enhancer element.

The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors.

pHIVTATA-CAT, a versatile vector to study transcriptional regulatory elements in mammalian cells.

We have constructed a vector, pHIVTATA-CAT, that contains the Escherichia coli cat gene, encoding chloramphenicol acetyltransferase, under the control of a minimal promoter consisting of the HIV TATA box and the adenovirus major late promoter initiator element. Putative transcriptional elements can be inserted either directly upstream from the TATA box or downstream from the reporter gene in an enhancer position. Transcription can be monitored enzymatically or by RNase protection mapping. An analysis of mRNAs generated from pHIVTATA-CAT constructs revealed that transcription starts at the transcription start point and no read-through transcripts are generated.

Identification of a functional cAMP response element in the secretogranin II gene.

Secretogranin II is an acidic secretory protein with a widespread distribution in secretory granules of neuronal and endocrine cells. The secretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels were increased in a neuronal cell line derived from the septal region of the brain and the level of secretogranin II gene expression was analysed. It was found that increased cAMP levels did, in fact, induce secretogranin II gene expression. To analyse the cis-acting sequence responsible for this induction, a hybrid gene containing the upstream region of the mouse secretogranin II gene fused to beta-globin as a reporter was constructed. Transfection analysis revealed that cAMP-induced transcription of the secretogranin II promoter/beta-globin gene in septal and insulinoma cells. DNA-protein binding assays showed that recombinant CRE-binding protein (CREB), produced in bacteria or human cells, bound in a sequence-specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma cells. An increase in the intracellular cAMP concentration activated a GAL4-CREB fusion protein upon transcription in neuroblastoma cells indicating the integrity of the cAMP signaling pathway to the nucleus. Basal as well as cAMP-stimulated transcription, directed from the secretogranin II promoter was, however, impaired in insulinoma cells by overexpression of CREB-2, a negative-acting CRE-binding protein. These results indicate that competitive effects are likely to occur between CRE-bound transcriptional activators and repressors. We conclude that cAMP-stimulated induction of secretogranin II gene transcription is mediated by the CRE motif in a cell-type-specific manner, and is likely to depend on the balance between positive and negative CRE-binding proteins in a particular cell type.

Neuron-specific gene expression of synapsin I. Major role of a negative regulatory mechanism.

The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. The human synapsin I gene was functionally analyzed to identify control elements directing the neuron-specific expression of synapsin I. By directly measuring the mRNA transcripts of a reporter gene, we demonstrate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between mouse and human. Deletion of a putative binding site for the zinc finger protein, neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST), abolished neuron-specific expression of the reporter gene almost entirely, allowing constitutively acting elements of the promoter to direct expression in a non-tissue-specific manner. These constitutive transcriptional elements are present as a bipartite enhancer, consisting of the region upstream (nucleotides -422 to -235) and downstream (nucleotides -199 to -143) of the putative NRSF/REST-binding site. The latter contains a motif identical to the cAMP response element. Both regions are not active or are only weakly active in promoting transcription on their own and show no tissue-specific preference. From these data we conclude that neuron-specific expression of synapsin I is accomplished by a negative regulatory mechanism via the NRSF/REST binding motif.

The human synapsin II gene promoter. Possible role for the transcription factor zif268/egr-1, polyoma enhancer activator 3, and AP2.

Synapsin II is a neuron-specific phosphoprotein that selectively binds to small synaptic vesicles in the presynaptic nerve terminal. Here we report the cloning and sequencing of the 5'-flanking region of the human synapsin II gene. This sequence is very GC-rich and lacks a TATA or CAAT box. Two major transcriptional start sites were mapped. A hybrid gene consisting of the Escherichia coli chloramphenicol acetyltransferase gene under the control of 837 base pairs of the synapsin II 5'-upstream region was transfected into neuronal and nonneuronal cells. While reporter gene expression was low in neuroblastoma and non-neuronal cells, high chloramphenicol acetyltransferase activities were monitored in PC12 pheochromocytoma cells. However, there was no correlation between reporter gene expression in the transfected cells and endogenous synapsin II immunoreactivity. Using DNA-protein binding assays we showed that the transcription factors zif268/egr-1, polyoma enhancer activator 3 (PEA3), and AP2 specifically contact the synapsin II promoter DNA in vitro. Moreover, the zif268/egr-1 protein as well as PEA3 were shown to stimulate transcription of a reporter gene containing synapsin II promoter sequences. In the nervous system, zif268/egr-1 functions as a "third messenger" with a potential role in synaptic plasticity. PEA3 is expressed in the brain and its activity is regulated by proteins encoded from non-nuclear oncogenes. We postulate that zif268/egr-1 and PEA3 couple extracellular signals to long-term responses by regulating synapsin II gene expression.

Regulation of synapsin I gene expression by the zinc finger transcription factor zif268/egr-1.

zif268/egr-1 is an immediate early response gene that is involved in regulation of growth and differentiation. Its mRNA encodes a sequence-specific transcriptional activator containing three zinc fingers that act as the DNA-binding domain. Although zif268/egr-1 is expressed in the nervous system during neuronal excitation, no target gene has yet been identified. Here we report that the zif268/egr-1 protein bound in vitro to two sites in the proximal regulatory region of the human synapsin I gene. The zif268/egr-1 protein was also shown to stimulate transcription from this control region in transactivation assays. Additionally, the presence of a putative neural-restrictive silencer element next to one of the zif268/egr-1-binding sites interfered with transactivation in a tissue-independent manner. An analysis of the temporal expression pattern of zif268/egr-1 and synapsin I during neuronal differentiation of P19 embryonal carcinoma cells revealed that zif268/egr-1 mRNA was induced on day 5 and synapsin I mRNA on day 8 after retinoic acid treatment. From this data we conclude that the synapsin I gene is a target of the zif268 transcription factor; however, intermediate factors may also be involved in the activation.