Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREBα and Δ isoforms and to an overexpression of the CREBβ isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREBα, β, and Δ) inactivated. In contrast to the CREBαΔ mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the αβ lineage, which is not affected in CREBαΔ mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the αβ T cell lineage. In contrast γδ T cell differentiation is normal in CREB mutant mice.

Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia

The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine.

The RIIβ regulatory subunit of protein kinase A binds to cAMP response element: An alternative cAMP signaling pathway

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIβ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIβ to a CRE was enhanced by cAMP, and in addition, RIIβ exhibited transcriptional activity as a Gal4-RIIβ fusion protein. These experiments identify RIIβ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

Cloning of inversion breakpoints in the Anopheles gambiae complex traces a transposable element at the inversion junction

Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd′ of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd′ suggests that the inversion may be the result of the transposable element’s activity. Characteristics of Odysseus’ terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.

Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI

We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac–Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.

Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes

Hepatic glucokinase plays a key role in glucose metabolism as underlined by the anomalies associated with glucokinase mutations and the consequences of tissue-specific knock-out. In the liver, glucokinase transcription is absolutely dependent on the presence of insulin. The cis-elements and trans-acting factors that mediate the insulin effect are presently unknown; this is also the case for most insulin-responsive genes. We have shown previously that the hepatic expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) is activated by insulin. We show here in primary cultures of hepatocytes that the adenovirus-mediated transduction of a dominant negative form of SREBP-1c inhibits the insulin effect on endogenous glucokinase expression. Conversely, in the absence of insulin, the adenovirus-mediated transduction of a dominant positive form of SREBP-1c overcomes the insulin dependency of glucokinase expression. Hepatic fatty acid synthase and Spot-14 are insulin/glucose-dependent genes. For this latter class of genes, the dominant positive form of SREBP-1c obviates the necessity for the presence of insulin, whereas glucose potentiates the effect of SREBP-1c on their expression. In addition, the insulin dependency of lipid accumulation in cultured hepatocytes is overcome by the dominant positive form of SREBP-1c. We propose that SREBP-1c is a major mediator of insulin action on hepatic gene expression and a key regulator of hepatic glucose/lipid metabolism.

A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells

Calbindin D28 encodes a calcium binding protein that is expressed in the cerebellum exclusively in Purkinje cells. We have used biolistic transfection of organotypic slices of P12 cerebellum to identify a 40-bp element from the calbindin promoter that is necessary and sufficient for Purkinje cell specific expression in this transient in situ assay. This element (PCE1) is also present in the calmodulin II promoter, which regulates expression of a second Purkinje cell Ca2+ binding protein. Expression of high levels of exogenous calbindin or calretinin decreased transcription mediated by PCE1 in Purkinje cells 2.5- to 3-fold, whereas the presence of 1 μM ionomycin in the extracellular medium increased expression. These results demonstrate that PCE1 is a component of a cell-specific and Ca2+-sensitive transcriptional regulatory mechanism that may play a key role in setting the Ca2+ buffering capacity of Purkinje cells.

Transcriptional regulation of the Xlim-1 gene by activin is mediated by an element in intron I

The Xlim-1 gene is activated in the late blastula stage of Xenopus embryogenesis in the mesoderm, and its RNA product becomes concentrated in the Spemann organizer at early gastrula stage. A major regulator of early expression of Xlim-1 is activin or an activin-like signal. We report experiments aiming to identify the activin response element in the Xlim-1 gene. The 5′ flanking region of the gene contains a constitutive promoter that is not activin responsive, whereas sequences in the first intron mediate repression of basal promoter activity and stimulation by activin. An intron-derived fragment of 212 nt is the smallest element that could mediate activin responsiveness. Nodal and act-Vg1, factors with signaling properties similar to activin, also stimulated Xlim-1 reporter constructs, whereas BMP-4 did not stimulate or repress the constructs. The mechanism of activin regulation of Xlim-1 and the sequence of the response element are distinct from activin response elements of other genes studied so far.

The small GTP-binding protein Rho links G protein-coupled receptors and Gα12 to the serum response element and to cellular transformation

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Gαs and Gαi2 families were found in human tumors; and members of the Gαq and Gα12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Gα12 family of heterotrimeric G protein α subunits strongly induced the SRE, while Gβ1γ2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Gα12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs

Histone mRNAs are naturally intronless and accumulate efficiently in the cytoplasm. To learn whether there are cis-acting sequences within histone genes that allow efficient cytoplasmic accumulation of RNAs, we made recombinant constructs in which sequences from the mouse H2a gene were cloned into a human β-globin cDNA. By using transient transfection and RNase protection analysis, we demonstrate here that a 100-bp sequence within the H2a coding region permits efficient cytoplasmic accumulation of the globin cDNA transcripts. We also show that this sequence appears to suppress splicing and can functionally replace Rev and the Rev-responsive element in the cytoplasmic accumulation of unspliced HIV-1-related mRNAs. Like the Rev-responsive element, this sequence acts in an orientation-dependent manner. We thus propose that the sequence identified here may be a member of the cis-acting elements that facilitate the cytoplasmic accumulation of naturally intronless gene transcripts.

Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1

The current studies explore the mechanism by which the sphingomyelin content of mammalian cells regulates transcription of genes encoding enzymes of cholesterol synthesis. Previous studies by others have shown that depletion of sphingomyelin by treatment with neutral sphingomyelinase causes a fraction of cellular cholesterol to translocate from the plasma membrane to the endoplasmic reticulum where it expands a regulatory pool that leads to down-regulation of cholesterol synthesis and up-regulation of cholesterol esterification. Here we show that sphingomyelinase treatment of cultured Chinese hamster ovary cells prevents the nuclear entry of sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound transcription factor required for transcription of several genes involved in the biosynthesis and uptake of cholesterol. Nuclear entry is blocked because sphingomyelinase treatment inhibits the proteolytic cleavage of SREBP-2 at site 1, thereby preventing release of the active NH2-terminal fragments from cell membranes. Sphingomyelinase treatment thus mimics the inhibitory effect on SREBP processing that occurs when exogenous sterols are added to cells. Sphingomyelinase treatment did not block site 1 proteolysis of SREBP-2 in 25-RA cells, a line of Chinese hamster ovary cells that is resistant to the suppressive effects of sterols, owing to an activating point mutation in the gene encoding SREBP cleavage-activating protein. In 25-RA cells, sphingomyelinase treatment also failed to down-regulate the mRNA for 3-hydroxy-3-methylglutaryl CoA synthase, a cholesterol biosynthetic enzyme whose transcription depends on the cleavage of SREBPs. Considered together with previous data, the current results indicate that cells regulate the balance between cholesterol and sphingomyelin content by regulating the proteolytic cleavage of SREBPs.

Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol

Caveolae form the terminus for a major pathway of intracellular free cholesterol (FC) transport. Caveolin mRNA levels in confluent human skin fibroblasts were up-regulated following increased uptake of low density lipoprotein (LDL) FC. The increase induced by FC was not associated with detectable change in mRNA stability, indicating that caveolin mRNA levels were mediated at the level of gene transcription. A total of 924 bp of 5′ flanking region of the caveolin gene were cloned and sequenced. The promoter sequence included three G+C-rich potential sterol regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequence. Deletional mutagenesis of individual SRE-like sequences indicated that of these two (at −646 and −395 bp) were essential for the increased transcription rates mediated by LDL-FC, whereas the third was inconsequential. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences, together with DNase I footprinting, confirmed nucleoprotein binding to the SRE-like elements as part of the transcriptional response to LDL-FC. A supershift obtained with antibody to SRE-binding protein 1 (SPEBP-1) indicated that this protein binds at −395 bp. There was no reaction at −395 bp with anti-Sp1 antibody nor with either antibody at −646 bp. The cysteine protease inhibitor N-acetyl-leu-leu-norleucinal (ALLN), which inhibits SREBP catabolism, superinhibited caveolin mRNA levels regardless of LDL-FC. This finding suggests that SREBP inhibits caveolin gene transcription in contrast to its stimulating effect on other promoters. The findings of this study are consistent with the postulated role for caveolin as a regulator of cellular FC homeostasis in quiescent peripheral cells, and the coordinate regulation by SREBP of FC influx and efflux.

Endoplasmic reticulum quality control of asialoglycoprotein receptor H2a involves a determinant for retention and not retrieval

The human asialoglycoprotein receptor H2a subunit contains a charged pentapeptide, EGHRG, in its ectodomain that is the only sequence absent from the H2b alternatively spliced variant. H2b exits the endoplasmic reticulum (ER) even when singly expressed, whereas H2a gives rise to a cleaved soluble secreted ectodomain fragment; uncleaved membrane-bound H2a molecules are completely retained and degraded in the ER. We have inserted the H2a pentapeptide into the sequence of the H1 subunit (H1i5), which caused complete ER retention but, unexpectedly, no degradation. This suggests that the pentapeptide is a determinant for ER retention not colocalizing in H2a with the determinant for degradation. The state of sugar chain processing and the ER localization of H1i5, which was unchanged at 15°C or after treatment with nocodazole, indicate ER retention and not retrieval from the cis-Golgi or the intermediate compartment. H1i5 folded similarly to H1, and both associated to calnexin. However, whereas H1 dissociated with a half time of 45 min, H1i5 remained bound to the chaperone for prolonged periods. The correct global folding of H2a and H1i5 and of other normal precursors and unassembled proteins and the true ER retention, and not exit and retrieval, suggest a difference in their quality control mechanism compared with that of misfolded proteins, which does involve retrieval. However, both pathways may involve calnexin.

A phylogenetic perspective on P transposable element evolution in Drosophila

The P element, originally described in Drosophila melanogaster, is one of the best-studied eukaryotic transposable elements. In an attempt to understand the evolutionary dynamics of the P element family, an extensive phylogenetic analysis of 239 partial P element sequences has been completed. These sequences were obtained from 40 species in the Drosophila subgenus Sophophora. The phylogeny of the P element family is examined in the context of a phylogeny of the species in which these elements are found. An interesting feature of many of the species examined is the coexistence in the same genome of P sequences belonging to two or more divergent subfamilies. In general, P elements in Drosophila have been transmitted vertically from generation to generation over evolutionary time. However, four unequivocal cases of horizontal transfer, in which the element was transferred between species, have been identified. In addition, the P element phylogeny is best explained in numerous instances by horizontal transfer at various times in the past. These observations suggest that, as with some other transposable elements, horizontal transfer may play an important role in the maintenance of P elements in natural populations.

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site. The central region of MBF1 (residues 35–113) is essential for the binding of FTZ-F1, MBF2, and TBP. When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBF1 did. Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription. These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human.