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Previous Article | Next Article
The Journal of Neuroscience, April 1, 2003, 23(7):2572
Upstream Stimulatory Factors Are Mediators of Ca2+-Responsive Transcription in Neurons
Wen G. Chen1, 2, Anne E. West2, Xu Tao2, Gabriel Corfas2, Marilyn N. Szentirmay3, Michèle Sawadogo3, Charles Vinson4, and Michael E. Greenberg1, 2 1 Program in Biological and Biomedical Sciences, Harvard Medical School, and 2 Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, 3 Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and 4 Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892
ABSTRACT
TOP
ABSTRACT
Introduction
To identify molecular mechanisms that control activity-dependent gene expression in the CNS, we have characterized the factors that mediate activity-dependent transcription of BDNF promoter III. We report the identification of a Ca2+-responsive E-box element, CaRE2, within BDNF promoter III that binds upstream stimulatory factors 1 and 2 (USF1/2) and show that USFs are required for the activation of CaRE2-dependent transcription from BDNF promoter III. We find that the transcriptional activity of the USFs is regulated by Ca2+-activated signaling pathways in neurons and that the USFs bind to the promoters of a number of neuronal activity-regulated genes in vivo. These results suggest a new function for the USFs in the regulation of activity-dependent transcription in neurons.
Key words: upstream stimulatory factor; USF1; USF2; activity-dependent transcription; brain-derived neurotrophic factor; BDNF; calcium; neural plasticity; activity-dependent neural development
Introduction
Neural activity arising from sensory input induces the expression of new gene products that contribute to enduring adaptations in the CNS. These activity-dependent changes include the refinement of cortical circuitry during development (Katz and Shatz, 1996
; Mao et al., 1999
7 subunit (nAchR
To identify molecular mechanisms that mediate activity-dependent gene expression in neurons, we have studied the transcriptional regulation of BDNF. BDNF is highly expressed in neurons and plays important roles in neuronal survival (Bonni et al., 1999
The cAMP/Ca2+-response element binding protein (CREB) is required for the activity-dependent transcription of a number of neuronal genes, including BDNF exon III (Shieh et al., 1998
Materials and Methods
Plasmids. BDNF pIII(170)-Luc, EF-
-gal, pSG424 (Gal4 only), Gal4-Luc, Gal4-USF2, pSG5-USF2, pSG5-USF2DN, 3×UBE-Luc (pU3ML-Luc), CRE-Luc, CMV-A-USF1, and the control vectors pML-Luc and CMV were described previously (Sheng et al., 1991
Cell culture, transfection, stimulation, and luciferase assay. Cortical neurons from embryonic day 18 (E18) Long-Evans rats (Charles River, Wilmington, MA) and E16 C57/Black 6 mice were cultured as described (Tao et al., 1998
Nuclear extracts and electrophoretic mobility shift assays. Nuclear extracts and electrophoretic mobility shift assays (EMSAs) were performed as described (Tao et al., 1998
5' exo
; New England Biolabs, Beverly, MA) with
Yeast one-hybrid screen. The yeast one-hybrid screen for CaRE2 binding proteins was performed by using the Matchmaker Yeast one-hybrid system (Clontech, Palo Alto, CA). Six repeats of the CaRE2 sequence (from nucleotides -56 to -39) were cloned upstream of the HIS3 gene. Then the plasmid was integrated into yeast genome to generate a yeast reporter strain, and the strain was transfected with a rat brain cDNA library containing ~1 × 106 independent clones fused to the transcriptional activation domain of the yeast Gal4 protein (Clontech). Colonies that grew on minimal medium lacking histidine were selected, and the cDNA-containing plasmids were recovered and sequenced from these colonies.
Immunocytochemistry. Anti-USF1 (C-20) and anti-USF2 (C-20) antibodies were purchased from Santa Cruz Biotechnology. Both antibodies were used at 1:400 for cell staining. The mouse TuJ1 anti-
Chromatin immunoprecipitation assay. E18 rat cortical neurons (2 × 107 cells) at 5 DIV were treated with 1% formaldehyde at room temperature for 20 min. After two washes with 1× PBS, the cross-linked neurons were scraped off the plates. The neurons were pelleted, resuspended in 200 µl of lysis buffer, and processed for chromatin immunoprecipitation with the chromatin immunoprecipitation (ChIP) assay kit (Upstate Biotechnology, Lake Placid, NY) with the following modifications: (1) the cell lysates were sheared by sonication for a total of 100 sec at 20 sec per interval; (2) the protein A agarose/antibody/transcription factor complex was washed in the low-salt immune complex wash buffer twice, high-salt immune complex wash buffer twice, LiCl immune complex wash buffer twice, and then 1× TE three times; and (3) the chromatin fragments that were pulled down with specific antibodies were resuspended in 200 µl of H2O, 10 µl of which was used for PCR for 28-30 cycles. Anti-Bad (N-20), anti-c-Myc (C-33), and anti-Id (Z-8) antibodies were purchased from Santa Cruz Biotechnology. PCR primer sequences for BDNF exon III were 5'-GCG CGG AAT TCT GAT TCT GGT AAT-3' and 5'-GAG AGG GCT CCA CGC TGC CTT GAC G-3'; for BDNF exon V were 5'-AAG TGT AAT CCC ATG GGT TAC ACG-3' and 5'-CAG GAA GTG TCT ATC CTT ATG AAC CG-3'; for COX-2 promoter were 5'-CCT GCC CCT ATG GGT ATT ATG C-3' and 5'-TTC GTG ACT GTG TCT TTC CGC-3'; for nAchR
Quantitative real-time RT-PCR. RNA was prepared with the Absolutely RNA kit (Stratagene). Total RNA (1 µg) was used for reverse transcription with the First Strand Superscript II kit (Invitrogen). PCR was performed in an iCycler (Bio-Rad, Hercules, CA) with the use of SYBR-green (PE Applied Biosystems, Foster City, CA). Each independent sample was assayed in triplicate. The threshold cycle for each sample was chosen from the linear range and converted to a starting quantity by interpolation from a standard curve run on the same plate for each set of primers. The firefly luciferase mRNA levels were normalized for each well to cotransfected
Results
An E-box sequence in BDNF promoter III is a Ca2+ response element
Previously, we found that 170 bp of the 5' flanking sequence of BDNF exon III is sufficient to activate reporter gene expression in response to membrane depolarization-induced Ca2+ influx via L-VSCCs in cultured embryonic rat cortical neurons (Tao et al., 1998
To characterize the element lying between CaRE1 and the CaRE3/CRE, we made two-nucleotide substitutions of the sequence between these elements in the context of the BDNF promoter III reporter gene construct. These mutant plasmids were transiently transfected into cultured cortical neurons, the cells were depolarized by exposure to elevated levels of KCl, and the induction of luciferase expression from the reporter gene was measured. As shown in Figure 1a, transcription from the wild-type BDNF pIII(170)-Luc reporter gene was induced significantly in response to membrane depolarization. Most mutations of the nucleotide sequence between -52 and -43 bp 5' to the BDNF exon III transcription initiation site severely reduced the ability of membrane depolarization to induce reporter gene expression, whereas mutations just outside this 10 bp region had little effect. These data identify the 10 bp nucleotide sequence from -52 to -43 bp relative to the BDNF exon III transcription initiation site as a critical Ca2+ response element that we have named CaRE2.
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Figure 1. CaRE2 is a Ca2+-responsive E-box element in BDNF promoter III. a, Characterization of CaRE2. A luciferase reporter plasmid driven by BDNF promoter III with either wild-type or mutant sequences was transfected into E18 + 3 DIV rat cortical neurons. The fold of induction equals the ratio of normalized luciferase activity from stimulated cells to that from the unstimulated neurons. The numbering indicates the position relative to the first transcription start site for BDNF exon III. In the mutant sequences the letters indicate the mutated bases, and the dashed lines represent unchanged nucleotides. b, Schematic diagram of Ca2+ response elements (CaREs) in BDNF promoter III. The CaRE2 sequence (bolded) from BDNF promoter III is shown and compared with the canonical E-box sequence. c, Characterization of the CaRE2 binding protein(s). Shown is the specific interaction of CaRE2 and its binding protein(s) in neurons. Nuclear extracts from P1 rat brain were mixed with radiolabeled probes containing the CaRE2 sequence before electrophoresis in a native polyacrylamide gel. Unbound radiolabeled CaRE2 probes (arrow II) migrated faster than the ones retarded by the CaRE2 binding protein(s) (arrow I). The specificity of the interaction was determined by the ability of excess unlabeled mutant or wild-type CaRE2-containing probes to compete away the radiolabeled CaRE2 probes from the DNA/protein complex. The sequences for the mutant probes (M1-M3, M6-M8) are shown in a.
Characterization and cloning of the CaRE2 binding protein
The core of the CaRE2 sequence (ATCATATGAC) fits the consensus for an E-box element (CANNTG). E-box elements are bound by members of the basic helix-loop-helix (bHLH) family of transcription factors. However, analysis of the TRANSFAC database (http://www.cbil.upenn.edu/tess/index.html) indicated that none of the previously characterized factors favor the specific E-box sequence we had identified as CaRE2 in BDNF promoter III. Therefore, to identify the transcription factor(s) that mediate Ca2+-dependent expression of BDNF exon III through CaRE2, we cloned and characterized the protein(s) that bind to this element.
Because BDNF is highly expressed in neurons, we asked whether there is a protein within the nucleus of cortical neurons that binds specifically to the CaRE2 sequence. Nuclear protein extracts were prepared from embryonic rat cortex, mixed with a radiolabeled probe encompassing the CaRE2 sequence, and then subjected to an EMSA. A protein in the cortical neuron nuclear extract was found to bind to and retard the mobility of the radiolabeled CaRE2 probe in a nondenaturing polyacrylamide gel (Fig. 1c). The association of this nuclear protein with CaRE2 was specific, because its binding to CaRE2 could be competed by the addition of an excess of unlabeled wild-type CaRE2 probe, but not with the addition of excess CaRE2 sequences that failed to support Ca2+-dependent induction in the context of the BDNF promoter III reporter gene (Fig. 1a). This correlation between the ability of the neuronal nuclear protein to bind CaRE2 sequences and the ability of these CaRE2 sequences to drive Ca2+-dependent transcription of BDNF promoter III supports the hypothesis that there exists a protein in cortical neurons for which the interaction with CaRE2 is required for Ca2+-dependent induction of BDNF promoter III transcription.
To identify the protein that regulates BDNF transcription through CaRE2, we used a yeast one-hybrid system to screen a rat brain cDNA library for CaRE2 binding protein(s). After screening 250,000 clones, we obtained three positive colonies. Protein extracts from these yeast contained a protein that bound to CaRE2 with the same specificity for wild-type and mutant CaRE2 sequences as the endogenous CaRE2 binding protein from neuronal nuclear extracts (data not shown). After sequencing of the brain cDNA recovered from these clones, we were surprised to find that in each case the expressed protein was the mammalian bHLH transcription factor USF1.
USF1 is a 43 kDa bHLH family transcription factor (Sawadogo et al., 1988
USF1 and USF2 bind CaRE2
To examine whether the USFs bind CaRE2, we first asked whether the USFs are part of the CaRE2 binding complex that we observed by EMSA with cortical neuron nuclear extracts. The addition of anti-USF1 or USF2 antibodies to the CaRE2-neuronal nuclear protein complex caused an additional retardation of the complex by EMSA, suggesting that both USF1 and USF2 are in the CaRE2-protein complex (Fig. 2a). In contrast, the addition of antibodies that recognize a closely related bHLH family transcription factor, c-Myc, had no effect on the CaRE2-protein complex. These results indicate that endogenous USF1 and USF2 are part of the complex of proteins present in neuronal extracts that bind to CaRE2.
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Figure 2. Upstream stimulatory factors (USFs) bind CaRE2. a, Endogenous USFs in neurons bind CaRE2. Nuclear extracts from P1 rat brain were incubated with excess unlabeled wild-type or mutant CaRE2 probes, anti-USF1 antibody, anti-USF2 antibody, or anti-c-Myc antibody on ice for 1 hr. Then radiolabeled CaRE2 probes were added to the mixture, which subsequently was subjected to EMSA. Arrow I marks the complex formed by the radiolabeled CaRE2 probes with the endogenous CaRE2 binding proteins. Arrow II indicates the supershifted complexes formed by anti-USF antibodies, the endogenous CaRE2 binding protein, and the radiolabeled CaRE2 probe. b, Recombinant USFs are sufficient to bind CaRE2. In vitro translated USF1 or USF2 proteins were mixed with radiolabeled CaRE2 probes and an excess of unlabeled wild-type or mutant CaRE2 probes and then subjected to EMSA. The sequences for the mutant CaRE2 probes are the same as those in Figure 1a. The arrow indicates the specifically retarded band.
To determine whether USF1 and USF2 are sufficient to bind to CaRE2 directly, we tested the ability of in vitro transcribed and translated USF1 and USF2 to bind to CaRE2 in an EMSA. As shown in Figure 2b, both USF1 and USF2 are capable of binding to CaRE2, as indicated by a retardation of the radiolabeled CaRE2 probe. Moreover, this binding shows the same specificity for CaRE2 sequences as we observed for the endogenous CaRE2 binding protein (Fig. 1c). This correlation between the ability of USFs to bind CaRE2 sequences and the ability of these sequences to support activity-dependent transcription from BDNF promoter III suggests that USF binding to CaRE2 is relevant for activity-dependent BDNF exon III transcription in neurons.
USFs functionally regulate CaRE2-dependent Ca2+-inducible BDNF exon III transcription
To determine whether the USFs mediate Ca2+-inducible BDNF exon III transcription, we asked whether dominant-negative versions of USF block the activity-dependent induction of BDNF promoter III reporter gene transcription. Both USF1 and USF2 are expressed constitutively in the nuclei of cultured embryonic cortical neurons (Fig. 3a-h). To disrupt their function, we used a deletion mutant of USF2 (DN-USF2) that lacks the N-terminal transcriptional activation domain and effectively competes with endogenous USF1 and USF2 for binding to promoter E-boxes (Qyang et al., 1999
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Figure 3. USFs mediate Ca2+-dependent activation of BDNF promoter III. a-h, USFs are expressed in the nucleus of cultured cortical neurons. E18 cortical cultures were grown for 5 DIV and then fixed and stained with the anti-USF1 or anti-USF2 antibodies (red; b, f) and an antibody against the neuronal marker
Both USF1 and USF2 can regulate activity-dependent BDNF exon III transcription
The dominant-negative experiments suggest that USFs are required for Ca2+ regulation of BDNF transcription. However, both dominant negatives inhibit USF1 and USF2 and therefore do not distinguish whether one family member or both are the key regulators of Ca2+-dependent BDNF transcription. To ask whether either USF1 or USF2 is sufficient for activity-dependent BDNF exon III expression or whether both are required for the response, we cultured embryonic cortical neurons from either USF1 or USF2 null mice or their wild-type or heterozygous siblings. These cells were transfected with the BDNF reporter construct, and the induction of luciferase in response to depolarization was measured. We observed no significant difference in the membrane depolarization-mediated induction of BDNF promoter III activity in any of the genotypes (Fig. 4a). Neither did we observe significant alteration in the Ca2+ responsiveness of BDNF promoter III in the USF1(
/
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Figure 4. Endogenous USF1 and USF2 regulate BDNF promoter III in vivo. a, USF 1 and USF2 are both capable of regulating the Ca2+-inducible activation of BDNF promoter III. E16 cortical neurons were obtained from USF1 and USF2 wild-type, heterozygous, or homozygous null mice and cultured in vitro for 3 d before transfection with the BDNF promoter III luciferase reporter. n represents the number of pups used for the measurement. The genotypes for the mice were determined by Southern blot analysis. Western blot analysis shows the relative amounts of USF1 and USF2 in nuclear extracts from USF1 and USF2 wild-type, heterozygous, and homozygous mice with antibodies specific to the USF1 and USF2 proteins. b, Endogenous USF1 and USF2 bind BDNF promoter III in vivo. Cultured E18 + 5 DIV rat cortical neurons were treated with formaldehyde to cross-link DNA binding proteins to chromatin and then were subjected to chromatin immunoprecipitation with antibodies specific to USF1 and USF2 or a number of control antibodies. After reversing cross-links, we subjected eluted genomic DNA fragments to PCR with primers specific to BDNF promoter III or BDNF exon V. One percent of the input of the sheared chromatin before immunoprecipitation was used as a positive control for the PCR reaction. Negative controls include antibodies against c-Myc (a transcription factor), Bad (a cytoplasmic protein), Id (a bHLH protein lacking a DNA binding domain), or beads only.
The experiments with USF null mice suggest that both USF1 and USF2 may regulate BDNF promoter III. To seek evidence that both USF1 and USF2 when expressed at physiological levels are bound to the endogenous CaRE2 element of BDNF promoter III in vivo, we used a ChIP assay to study the in vivo occupancy of BDNF promoter III by USF1 and USF2. After first cross-linking DNA-bound proteins to chromatin in neuronal cultures, we lysed the cells and sheared the chromatin to an average of 150 bp in length. Next we used specific antibodies against USF1 and USF2 to immunoprecipitate these proteins along with the bound chromatin. After extensive washing and reversal of the cross-linking, we used specific PCR primers to test for the presence of BDNF promoter sequences that coimmunoprecipitated with the USF proteins. We were able to detect BDNF promoter III sequences in the USF immunoprecipitates (Fig. 4b). However, BDNF exon V, which should have been sheared away from promoter III, was not found in the pellet, indicating that immunoprecipitation of promoter III was specific (Fig. 4b). To control for antibody specificity, we performed the immunoprecipitation with a number of control antibodies and found that neither promoter III nor exon V of the BDNF gene immunoprecipitated with any of the control antibodies (Fig. 4b), although the anti-c-Myc antibody can precipitate its target promoters effectively in NIH 3T3 cells (data not shown). In total, these data strongly suggest that both USF1 and USF2 regulate transcription from promoter III of the endogenous BDNF gene in vivo.
USFs are activated by Ca2+ signals via L-VSCCs
The ability of USFs to regulate transcription of BDNF exon III through a Ca2+-responsive element suggested to us that the transcriptional activity of USFs might be regulated by Ca2+-activated signaling pathways in neurons. To isolate the activity of the USFs from that of other Ca2+-responsive transcription factors on BDNF promoter III, we studied the effects of calcium signaling pathways on transcription from a plasmid containing three copies of a consensus USF binding element in front of a luciferase reporter gene (3×UBE-Luc) (Qyang et al., 1999
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Figure 5. USFs are Ca2+-regulated transcription factors activated via L-type VSCCs. a, USF binding element (UBE) is regulated by membrane depolarization. Cultured E18 + 3 DIV rat cortical neurons were transfected with either a firefly luciferase reporter gene driven by three repeats of the consensus USF binding element (3×UBE) or the control vector. b, c, USFs are activated by Ca2+ influx via L-type VSCC. Cultured E18 + 3 DIV rat cortical neurons were transfected with either the 3×UBE-Luc reporter plasmid or CRE-Luc plasmids. At 2 d after transfection the cells were treated with nimodipine, APV, or the carrier solution before depolarization with 50 mM KCl or glutamate stimulation. After 8 hr of stimulation the cells were lysed, and the luciferase activities were measured. d, e, The transcriptional activity of USF2 is regulated by membrane depolarization. Cultured E18 + 3 DIV rat cortical neurons were transfected with a Gal4-luciferase reporter plasmid and either an expression plasmid for the Gal4 DNA binding domain alone (control) or the Gal4-DNA binding domain fused to the transcriptional activation domain of USF2. d, A renilla luciferase reporter plasmid was cotransfected as a control for transfection efficiency and sample handling. At 2 d after transfection the cells were depolarized with 50 mM KCl for 9 hr; then the cells were lysed, and the luciferase activities were measured. e, An
There are a number of mechanisms by which Ca2+ influx could regulate the USFs. In mast cells surface receptor activation leads to nuclear translocation of USF2 (Frenkel et al., 1998
Because Ca2+ influx does not appear to regulate the nuclear localization or DNA binding of the USFs, we considered the possibility that Ca2+-dependent signaling pathways might regulate the transcriptional activation domains of the USFs directly. To determine whether Ca2+ influx directly regulates the ability of the USFs to activate transcription, we tethered the transcriptional activation domain of USF2 to the DNA binding domain of the yeast transcription factor Gal4 (Luo and Sawadogo, 1996b
USFs are general regulators of activity-dependent transcription
These experiments suggest that USFs are Ca2+-regulated transcription factors in neurons and raise the possibility that USFs may contribute to the inducible expression of activity-regulated genes in addition to BDNF. In fact, a number of genes characterized in vitro by EMSA and by reporter gene assays as USF target genes (Paterson et al., 1995
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Figure 6. USFs are general regulators of Ca2+-dependent gene transcription in the nervous system. a, USFs bind to the promoters of several activity-regulated neuronal genes in vivo. Cultured E18 + 5 DIV rat cortical neurons were treated with formaldehyde to cross-link DNA binding proteins to chromatin and were subjected to chromatin immunoprecipitation with antibodies specific to USF1 and USF2 or a number of control antibodies. After reversing the cross-linking, we subjected eluted genomic DNA fragments to PCR with primers specific for COX-2, Nur77, nAchR
Discussion
USFs regulate activity-dependent transcription from BDNF promoter III
In this study we have defined a new Ca2+-responsive E-box element, CaRE2 (ATCATATGAC), in BDNF promoter III. This element is required to confer Ca2+-responsive transcriptional activation of BDNF promoter III. Using a yeast one-hybrid screen, we identified the upstream stimulatory factors (USF1 and USF2) as CaRE2 binding proteins. Several lines of evidence indicate that the USFs are relevant regulators of activity-dependent BDNF transcription through CaRE2. We have shown that endogenous USF1 and USF2 from neuronal nuclear extracts bind to the CaRE2 sequence in vitro. The USFs are sufficient to bind directly to the CaRE2 sequence and only bind CaRE2 sequences that support activity-dependent transcription of the BDNF promoter III luciferase reporter. Overexpression of dominant-negative forms of the USFs block activity-dependent transcription from BDNF promoter III, suggesting that the transcriptional activity of the USFs is required for this induction. Finally, using a chromatin immunoprecipitation assay, we demonstrate that in neurons both USF1 and USF2 are bound to the CaRE2-containing region of promoter III in the endogenous BDNF gene. Either USF1 or USF2 appears to be sufficient to support activity-dependent BDNF expression, because we observe that depolarization-induced increases in BDNF promoter III activity occur normally in mice null for either USF1 or USF2, as well as in USF1(
A new role for the USFs in the CNS
Although the USFs were among the first bHLH transcription factors to be identified and they were shown to be expressed in brain (Sirito et al., 1994
On BDNF promoter III we have shown that the USFs cooperate with other transcription factors to regulate activity-dependent gene expression. We have identified three discrete Ca2+-response elements (CaRE1, CaRE2, and CaRE3/CRE) in BDNF promoter III that are required for the induction of exon III transcription in response to Ca2+ influx in neurons (Tao et al., 1998
One possibility is that each of the BDNF regulatory factors serves a unique but essential role in regulating Ca2+ induction of BDNF transcription. Like the induction of BDNF exon III expression itself, the activity of the CaRE1 binding protein CaRF is regulated in a Ca2+- and neural-selective manner, suggesting that this factor may confer stimulus and cell type selectivity with the expression of BDNF exon III (Tao et al., 2002
Interestingly, USF binding elements are found in tandem with CREB binding elements in a number of promoters (Cvekl et al., 1994
Regulation of USF transcriptional activity
In addition to showing that CaRE2 is required for activity-dependent induction of BDNF promoter III, our findings suggest that the activity of USFs may be induced by calcium influx into neurons. As shown in Figure 5, membrane depolarization induced robust activation of USF-dependent transcription. This effect was blocked completely by L-type VSCCs-specific inhibitors but was unaffected by NMDA receptor inhibitors, suggesting that Ca2+ entry via L-type VSCCs mediates the activation of USF-dependent transcription in membrane-depolarized neurons. In contrast to membrane depolarization with elevated levels of KCl, the addition of glutamate to cultured neurons failed to activate USF-dependent transcription. Under these conditions glutamate induces significant Ca2+ influx through NMDA receptors and a modest amount of calcium influx through the L-type VSCC (Bading et al., 1995
Because the transcriptional activity of a Gal4-USF2 fusion protein also can be regulated rapidly by calcium signaling pathways in neurons, we postulate that a post-translational modification of either USFs or components of the transcriptional machinery that USFs bring to the promoters of Ca2+-responsive genes may mediate Ca2+ induction of USF-dependent transcription. Phosphorylation is a common means of regulating the activity of transcription factors, and USF1 has been reported to be a phosphoprotein in HeLa cells (Galibert et al., 1997
One mechanism by which neuronal activity might control the function of the USFs is by regulating the interaction between USFs and components of the basal transcription machinery. The USFs have been shown to interact physically with TAFII55, and via this interaction USFs can recruit the TATA-box binding complex TFIID (Workman et al., 1990
FOOTNOTES Received Aug. 26, 2002; revised Nov. 15, 2002; accepted Jan. 13, 2003.
We acknowledge the gracious support of the F. M. Kirby Foundation to the Division of Neuroscience (M.E.G.), Mental Retardation Research Center Grant HD18655 (M.E.G.), and National Institutes of Health Grants NS28829-07 (M.E.G.) and CA79579 (M.S.). This work also was supported by a Howard Hughes Medical Institute Predoctoral Fellowship (W.G.C.) and an American Cancer Society Postdoctoral Fellowship (A.E.W.). We thank Dr. Mario Sirito for help and advice on the USF knock-out mice and Pieter Dikkes and Ruth Ann-Pimental for technical assistance. We also thank Dr. Eric Griffith, Dr. Chris Cowan, Dr. Ricardo Dolmetsch, Dr. Janine Zieg, Paul L. Greer, Elizabeth Nigh, and members of the Greenberg laboratory for helpful discussions and critical reading of this manuscript.
Correspondence should be addressed to Dr. Michael E. Greenberg, Division of Neuroscience, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115. E-mail: michael.greenberg{at}tch.harvard.edu.
X. Tao's present address: Curis Incorporated, Cambridge, MA 02138.
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