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. 2010 Feb 8:11:12.
doi: 10.1186/1471-2156-11-12.

Sp1 and KLF15 regulate basal transcription of the human LRP5 gene

Jiangxia Li  1 , Yang Yang, Baichun Jiang, Xiyu Zhang, Yongxin Zou, Yaoqin Gong
Affiliations

Sp1 and KLF15 regulate basal transcription of the human LRP5 gene

Jiangxia Li et al. BMC Genet. .

Abstract

Background: LRP5, a member of the low density lipoprotein receptor superfamily, regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in adult organisms. However, how the expression of human LRP5 gene is regulated remains unclear.

Results: In order to characterize the transcriptional regulation of human LRP5 gene, we cloned the 5' flanking region and evaluated its transcriptional activity in a luciferase reporter system. We demonstrated that both KLF15 and Sp1 binding sites between -72 bp and -53 bp contribute to the transcriptional activation of human LRP5 promoter. Chromatin immunoprecipitation assay demonstrated that the ubiquitous transcription factors KLF15 and Sp1 bind to this region. Using Drosophila SL2 cells, we showed that KLF15 and Sp1 trans-activated the LRP5 promoter in a manner dependent on the presence of Sp1-binding and KLF15-binding motifs.

Conclusions: Both KLF15 and Sp1 binding sites contribute to the basal activity of human LRP5 promoter. This study provides the first insight into the mechanisms by which transcription of human LRP5 gene is regulated.

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Figures

Figure 1
Figure 1
Identification of the TSS of the LRP5 gene and the putative transcription factor binding sites in human LRP5 promoter. (A) The transcription start site (TSS) was determined by primer extension, and the extended product is indicated by the arrow. (B) Nucleotide sequence surrounding the 5' end of human LRP5. The numbering of the sequence is relative to TSS. Distinct DNA binding sites for transcription factors are indicated (underlined). The numbers above the sequence referred to the 5' position of serial deletion constructs. The primers used in ChIP assay are indicated in italic and bold.
Figure 2
Figure 2
A sequence located between -72 and -53 confers basal transcriptional activity of human LRP5 promoter. (A and B) Serial 5' deletion constructs were transfected into U2OS and HEK293 cells, and the relative luciferase activity was determined. The activity of pGL3-basic construct in A or that of pWT-359 construct in B was arbitrarily set to 100%, and the relative luciferase activity of the other constructs was calculated accordingly. Each bar represents the value of mean ± SEM.
Figure 3
Figure 3
KLF15 and Sp1 binding sites located between -72 and -53 contribute to the basal transcriptional activity of human LRP5 promoter. (A) Schematic representation of the KLF15 and Sp1 elements. Point mutations (underlined) were introduced to change the binding sites. (B) Constructs with native (pWT-72) or mutant KLF15 and/or Sp1 sites were transfected into HEK293 and U2OS cells, and the luciferase activity was determined. The luciferase activity of pWT-72 was arbitrarily set to 100%, and the activities of other constructs were calculated accordingly. (C and D) Sp1 and KLF15 transactivated the LRP5 promoter only if Sp1 and KLF15 binding sites were present. SL2 cells were cotransfected with either wild type construct (pWT-72) or mutated constructs (pMT1-72 or pMT2-72) along with the Sp1 (3C) or KLF15 (3D) expression constructs, and the relative luciferase activity was determined. The luciferase activity cotransfected with control vector (empty vector, EV) was set to 100%, and the relative activity under KLF15 or Sp1 stimulation was calculated accordingly. (E) Chromatin immunoprecipitation assay of KLF15 and Sp1 binding to human LRP5 promoter. The bindings of KLF15 and Sp1 to the human HSD17B5 promoter were used as a positive control (upper panel). The bindings of KLF15 to the LRP5 promoter (lower right panel) and the binding of Sp1 to the LRP5 promoter (lower left panel) were determined by ChIP using anti-KLF15 and anti-Sp1 antibodies, respectively. Anti-IgG antibodies were used as a negative control. The associated chromatin DNA fragments were amplified with the primer pairs flanking the Sp1 and KLF15 binding sites. Chromatin DNA input as described in the material and methods was subjected to the same PCR amplification.
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