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. 2012 Sep 1;17(4):571–585. doi: 10.2478/s11658-012-0028-1

Cloning of the human activated leukocyte cell adhesion molecule promoter and identification of its tissue-independent transcriptional activation by Sp1

Fang Tan 1, Flaubert Mbunkui 2, Solomon F Ofori-Acquah 1,3,✉
PMCID: PMC3683579  NIHMSID: NIHMS472655  PMID: 22941204

Abstract

Activated leukocyte cell adhesion molecule (ALCAM) belongs to the immunoglobulin cell adhesion molecule super family. ALCAM is implicated in tumor progression, inflammation, and the differentiation of hematopoietic stem cells. Hitherto, the identity of regulatory DNA elements and cognate transcription factors responsible for ALCAM gene expression remained unknown. In this report, the human ALCAM promoter was cloned and its transcriptional mechanisms elucidated. The promoter is TATA-less and contains multiple GC-boxes. A proximal 650-bp promoter fragment conferred tissue-independent activation, whereas two contiguous regions upstream of this region negatively influenced promoter activity in a tissue-specific manner. The positive regulatory promoter region was mapped to a core 50 base pair sequence containing a conical Sp1 element. Mutation analysis revealed that this element alone or in tandem with elements immediately upstream was required for maximal promoter activity. Chromatin analysis revealed that Sp1 binds exclusively to the canonical binding sequence in vivo, but not to DNA sequence immediately upstream. Finally, we showed that over-expression of Sp1 significantly increased the basal promoter activity. Thus, Sp1 activated the ALCAM promoter in most cells. These findings have important ramifications for unraveling the roles of ALCAM in inflammation and tumorigenesis.

Key words: ALCAM, Cis elements, Endothelial cells, Epithelial cells, Hematopoietic cells, Promoter activity, Sp1, Transcriptional regulation

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Abbreviations used

ALCAM

activated leukocyte cell adhesion molecule

CHIP

chromatin immunoprecipitation

EMSA

electrophoretic mobility shift assay

PAECs

pulmon. artery endothelial cells

PMVECs

pulmon. micro-vascular endothelial cells

pN3Sp1

plasmid DNA expressing Sp1

Sp1

specificity protein 1

TBP

TATA-box binding protein

TFIIB

transcription factor IIB

TRANSFAC

TRANScription FACtor database

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