Involvement of the GC-rich sequence and specific proteins (Sp1/Sp3) in the basal transcription activity of neurogranin gene

doi:10.1016/j.bbrc.2006.04.054

Biochemical and Biophysical Research Communications

Volume 345, Issue 1, 23 June 2006, Pages 124-132

https://doi.org/10.1016/j.bbrc.2006.04.054 Get rights and content

Abstract

Neurogranin (Ng), a neuronal protein implicated in learning and memory, contains a TATA-less promoter. Analysis of 5′-deletion mutations and site-directed mutations of the mouse Ng promoter revealed that a 258 bp 5′-flanking sequence (+3 to +260) conferred the basal transcription activity, and that the GC-rich sequence (+22 to +33) served as an important determinant of the promoter activity. Transient transfection of the Sp1 expression plasmid transactivated the reporter activity in neuroblastoma N2A cells while knocking down of endogenous Sp1 expression resulted in a 2.5-fold reduction of the reporter activity in HEK 293 cells. Exogenous expression of Sp3 in HEK 293 cells, however, repressed the reporter activity by 50%. Nevertheless, by gel shift assays, Sp1 and Sp3 were not found to be responsible for the protein-DNA complexes formed by the GC-rich sequence. Moreover, a nuclear factor from the mouse brain tissues was discovered to bind to multiple AT-rich regions in Ng promoter.

Section snippets

Materials and methods

Cell culture. Human embryonic kidney 293 (HEK 293), neuroblastoma Neuro-2a (N2A) (ATCC, Rockville, MD), and hypothalamic GT1-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 25 mM Hepes, 3.7 g/L NaH₂CO₃, 100 U/ml penicillin, and 100 μg/ml streptomycin, with a final pH of 7.3–7.4. Cells were cultured in a humidified incubator with 5% CO₂ at 37 °C and frequently checked for mycoplasma

The GC-rich sequence (+22 to +33) in the 5′-flanking sequence is an important determinant in the basal transcription activity of the mouse Ng gene

To characterize the mouse Ng promoter, a 2.2 kb sequence corresponding to the 5′-flanking sequence of the mouse Ng gene was amplified by PCR. Luciferase reporter plasmids containing a series of 5′-deletion mutations of Ng promoter were constructed and transfected to HEK 293 cells. Promoter analysis showed that a 258 bp 5′-flanking sequence of the mouse Ng gene that encompasses a region from +3 to +260 conferred the full promoter activity. This construct showed similar reporter activity as that

Acknowledgments

The authors thank Dr. James T. Kadonaga for pSp1-778C plasmid, Dr. Guntram Suske for pN3-Sp3FL-new plasmid and pN3 vector, and Dr. Pamela Mellon for GT1-7 cells. This work was supported in part by a grant from the Ministry of Education Singapore via the University Research Council of National University of Singapore R-154-000-228-112, and by Academic Research Grant R-398-000-024-112 to F.-S.S.

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These results suggested that PS induced impairment of spatial learning and memory in offspring animals might be associated with the activated hypothalamic–pituitary–adrenal (HPA) axis and the oxidative damage to DNA in the hippocampus. Ng gene promoter contains putative binding sites for transcription factors such as AP-1, AP-2, SP1 and NF, which are likely responsible for conferring basal transcriptional activity (Gui et al., 2006; Sato et al., 1995). Ng gene has the cis-acting elements, such as steroid hormones (glucocorticoid/ progesterone) acceptor response element (GRE) (Iniguez et al., 1993).
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LRP16 gene has been characterized as an estrogen-responsive gene. One 1/2ERE/GC-rich site was previously identified to be indispensable for −676/−214 (region A) fragment within LRP16 regulatory region to confer E2 action. Here, we report that −213/−24 fragment (region B) has higher E2-responsiveness than that of region A in MCF-7 cells, but not in HeLa cells. Deletion and mutation analyses of region B showed that multiple GC-sites are involved in the E2-stimulated response and one 30-bp fragment (−213 to −184 bp) is essential for conferring maximum E2-responsiveness. Results from the cotransfection assays containing Sp1-siRNA revealed that Sp1 is required for the basal transcription activity and E2-responsiveness of both regions A and B. Northern blot analysis demonstrated that inhibition of Sp1 in MCF-7 cells not only decreased the basal expression of LRP16, but markedly impaired its upregulation by E2. Results from gel mobility shift assays exhibited the direct binding of Sp1 protein to the 28-bp fragment (−211 to −184 bp), which was enhanced by the ERα titer. Moreover, the functional interaction of ERα and Sp1 proteins in the presence of E2 at the GC-rich sites in region B was confirmed by chromatin immunoprecipitation (ChIP) assays. In general, these results demonstrate that GC-rich sites in the proximal promoter of LRP16 gene are sufficient for E2 activation of LRP16 and the −213/−184 fragment containing only one GC site is essential for the maximal induction in MCF-7 cells. We also provide a model for Sp1-dependent regulation of genes by E2 through GC-rich motifs.
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Sp1 is known to play a role in the regulation of genes lacking a functional TATA box. Examples of this are the neurogranin gene and myeloid Elf-1-like factor, each of which has a TATA-less promoter region in which Sp1 binds to a GC-box to activate transcription [18,19]. The presence of numerous Sp1 binding sites in the promoter region of survivin suggested that Sp1 might be involved in the regulation of survivin activity.
Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. In this study, we cloned and identified the proximal 269 bp promoter of survivin gene, which exhibited strong promoter activity in HeLa cells. The TATA-less, GC-rich promoter contains 7 putative binding sites for Sp1, two of which (one at position −148 to −153, the other at position −127 to −140) are essential in regulating basal survivin promoter activity. Not only Sp1 but also Sp3 can activate the survivin promoter, which were proven by EMSA, blocking Sp1 or Sp3 using RNAi or mithramycin treatment of HeLa cells, and overexpression of Sp1 or Sp3. Our results collectively suggest that Sp1 cooperates with Sp3 to regulate survivin promoter activity.
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Involvement of the GC-rich sequence and specific proteins (Sp1/Sp3) in the basal transcription activity of neurogranin gene

Abstract

Section snippets

Materials and methods

The GC-rich sequence (+22 to +33) in the 5′-flanking sequence is an important determinant in the basal transcription activity of the mouse Ng gene

Acknowledgments

J. Biol. Chem.

Arch. Biochem. Biophys.

Brain Res.

Behav. Brain Res.

Brain Res. Mol. Brain Res.

Neurosci. Lett.

Neurobiol. Aging

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Gene

Cell

J. Biol. Chem.

Gene

J. Biol. Chem.

J. Biol. Chem.

Homeostatic tuning of Ca2+ signal transduction by members of the calpacitin protein family

J. Neurosci. Res.