Sp1 and CREB regulate basal transcription of the human SNF2L gene

https://doi.org/10.1016/j.bbrc.2008.01.111Get rights and content

Abstract

Imitation Switch (ISWI) is a member of the SWI2/SNF2 superfamily of ATP-dependent chromatin remodelers, which are involved in multiple nuclear functions, including transcriptional regulation, replication, and chromatin assembly. Mammalian genomes encode two ISWI orthologs, SNF2H and SNF2L. In order to clarify the molecular mechanisms governing the expression of human SNF2L gene, we functionally examined the transcriptional regulation of human SNF2L promoter. Reporter gene assays demonstrated that the minimal SNF2L promoter was located between positions −152 to −86 relative to the transcription start site. In this region we have identified a cAMP-response element (CRE) located at −99 to −92 and a Sp1-binding site at −145 to −135 that play a critical role in regulating basal activity of human SNF2L gene, which were proven by deletion and mutation of specific binding sites, EMSA, and down-regulating Sp1 and CREB via RNAi. This study provides the first insight into the mechanisms that control basal expression of human SNF2L gene.

Section snippets

Materials and methods

Cell culture. The rat pheochromocytoma PC12 cells were cultured on collagen coated dishes in RPMI 1640 medium supplemented with 15% horse serum, 2.5% fetal calf serum and antibiotics. HeLa cells and C6 cells were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum and antibiotics. Cells were incubated in the humidified incubator equilibrated with 5% CO2 at 37 °C and passaged using standard cell culture techniques.

Plasmids and constructs. A 855 bp fragment

Identification of the putative SNF2L promoter region

To isolate the human SNF2L promoter we performed a BLAST search of human genome sequence using the human SNF2L cDNA (GenBank Accession No. NM_003069) as a query. The 5′ sequence of the SNF2L cDNA perfectly matched to a genomic sequence (GenBank Accession No. NW_927721). Computer-based analysis of this region with MatInspector software failed to find any canonical TATA box or CAAT-like sequence. However, several potential transcription factor binding motifs were identified in the promoter region

Acknowledgments

This work was supported by National Basic Research Program of China (973 Program) Grant 2007CB512001 and National Science Fund Grant 30671163.

Cited by (5)

  • Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells

    2014, Gene
    Citation Excerpt :

    The importance of SP1 and CRE binding sites to galanin gene expression has been shown for the bovine galanin gene in induced and non-induced processes, as discussed earlier. Their relevance is also supported by the observation that interactions among SP1 and CRE binding sites have been reported in the literature for other promoters and may be a common signature that drives the expression of several genes (Piera-Velazquez et al., 2007; Xia et al., 2008). It is possible that the same mechanism may operate to regulate mouse galanin in ES cells.

  • Characterization of nuclear localization signal in the N terminus of CUL4B and its essential role in cyclin E degradation and cell cycle progression

    2009, Journal of Biological Chemistry
    Citation Excerpt :

    Whole cell lysates and cytoplasmic and nuclear extracts were quantified using a BCA protein assay kit (Beyotime). Western blotting was performed as described previously (20). In brief, equal amounts (30–60 μg) of extracts were subjected to 12% SDS-polyacrylamide gel for electrophoresis, transferred to a polyvinylidene difluoride membrane (Amersham Biosciences), followed by incubation with specific primary antibodies at the appropriate dilution overnight at 4 °C.

View full text