Yoav Citri

A family of unusually spliced biologically active transcripts encoded by a Drosophila clock gene

Complementary DNA cloning of the transcripts of the Drosophila clock gene period reveals three distinct transcripts. These result from unusual splicing pathways, one involving a CG 3′ splice site and one resulting in the use of two different reading frames in one exon, and they predict three separate proteins. Two of the cloned cDNAs can restore clock function to mutant arrhythmic flies.

Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms

Long-term plasticity of the central nervous system (CNS) involves induction of a set of genes whose identity is incompletely characterized. To identify candidate plasticity-related genes (CPGs), we conducted an exhaustive screen for genes that undergo induction or downregulation in the hippocampus dentate gyrus (DG) following animal treatment with the potent glutamate analog, kainate. The screen yielded 362 upregulated CPGs and 41 downregulated transcripts (dCPGs). Of these, 66 CPGs and 5 dCPGs are known genes that encode for a variety of signal transduction proteins, transcription factors, and structural proteins. Seven novel CPGs predict the following putative functions:cpg2—a dystrophin-like cytoskeletal protein;cpg4—a heat-shock protein:cpg16—a protein kinase;cpg20—a transcription factor;cpg21—a dual-specificity MAP-kinase phosphatase; andcpg30 andcpg38—two new seven-transmembrane domain receptors. Experiments performed in vitro and with cultured hippocampal cells confirmed the ability of thecpg-21 product to inactivate the MAP-kinase. To test relevance to neural plasticity, 66 CPGs were tested for induction by stimuli producing long-term potentiation (LTP). Approximately one-fourth of the genes examined were upregulated by LTP. These results indicate that an extensive genetic response is induced in mammalian brain after glutamate receptor activation, and imply that a significant proportion of this activity is coinduced by LTP. Based on the identified CPGs, it is conceivable that multiple cellular mechanisms underlie long-term plasticity of the nervous system.

A brain-specific transcription activator

We have identified a DNA binding protein, named BETA, that interacts with the same (B) transcriptional regulatory sequence as the known transcription factor NF-kappa B. BETA is found only in gray matter throughout the brain, and not in a variety of other rat tissues. Two binding sites for BETA are present adjacent to the promoter of the rat proenkephalin gene. Transfection of primary brain cultures that express BETA, with a reporter gene driven by the SV40 promoter linked to BETA DNA binding sites, results in transcriptional activation. We infer that BETA is a brain-specific transcription activator.

NF-kappa B activates the HIV promoter in neurons.

Human immunodeficiency virus (HIV) infection of the brain leads to massive neuronal damage, resulting in the AIDS (acquired immunodeficiency syndrome) dementia complex (ADC). A recent study using transgenic mice indicates that neurons possess transcription factors capable of activating the HIV promoter. To identify these, we transfected two types of primary cultures of rat neurons with HIV promoter‐reporter gene constructs. The two kappa B regulatory sites in the HIV long terminal repeat (LTR) are shown to be essential for strong promoter activity. Two proteins present in neurons, BETA and an NF‐kappa B‐like protein, can bind the kappa B sites. These proteins are shown to belong to distinct families of transcription factors. Mutation analysis and transfection of a dominant negative NF‐kappa B mutant, indicate that the neuronal NF‐kappa B‐like activity mediates HIV promoter activation. cDNA cloning, biochemical and immunological analyses indicate that neuronal NF‐kappa B is similar to NF‐kappa B of other tissues. Transfections of primary neuron cultures with an HIV promoter‐beta‐galactosidase construct show that within these cultures, neurons are indeed the cells that highly activate the HIV promoter. Thus, analogous to the situation in T‐lymphocytes and macrophages, NF‐kappa B is an activator of HIV transcription in neurons.

Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes.

Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered.

CPG16, a Novel Protein Serine/Threonine Kinase Downstream of cAMP-dependent Protein Kinase

Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested,e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase.