An L1 element disrupts human bone sialoprotein promoter: lack of tissue-specific regulation by distalless5 (Dlx5) and runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements.
Bone sialoprotein (BSP) is a phosphorylated and sulphated glycoprotein with hydroxyapatite nucleating properties that is specifically expressed in association with physiological and pathological mineralization. Although previous studies have indicated that tissue-specific expression of murine BSP is regulated through a proximal homeodomain element that binds distalless5 (Dlx5) transcription analysis of the homologous human promoter revealed modest enhancement in osteogenic cells. Moreover, whereas forced expression of an antisense Dlx5 vector increased transcription, Dlx5 expression did not alter transcription significantly. Since extended promoter sequences are required to confer absolute tissue-specific expression of BSP in vivo, we characterized the upstream region of the human BSP gene. In contrast to the rat and mouse promoters, which show conserved sequences extending several kbs upstream, analysis of approximately 3 kb of the human promoter showed no sequence conservation beyond -0.99 kb. Southern blot analysis of genomic DNA from four different BAC clones showed that this sequence was not an aberration in the human genomic library used to isolate the BSP gene. Using clone BAC H-NH0811I08, the human BSP promoter sequence was extended approximately 8 kb upstream from which the non-homologous region was characterized as a 3.48 kb insert coding for an L1 retrotransposon element. Transcriptional analyses of chimeric promoter constructs revealed that the retrotransposon element suppresses transcription <80%. Upstream of the inserted DNA several regions, varying in length from 26 to 161 bps, were conserved within the mouse and human promoters. One of these conserved regions included a runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements consensus element in reverse orientation. Whereas a multimeric form of the element was transcriptionally active in response to Runx2/Cbfa1 when ligated to the BSP basal promoter, the single element in the context of the extended promoter was unresponsive. These studies have characterized the upstream promoter of the human BSP gene, which is interrupted by a unique high-frequency DNA insert that suppresses BSP gene transcription.
Kiyoshima
Tamotsu
T
CIHR Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Room 239 FitzGerald Building, 150 College Street, Toronto, Ontario M5S 3E2, Canada.
Yamauchi
Masato
M
Wong
Cherrie
C
Jheon
Andrew
A
Ganss
Bernhard
B
Sodek
Jaro
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D009363:Neoplasm Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D050659:Core Binding Factor Alpha 1 Subunit; C100596:DLX5 protein, human; C501120:Dlx5 protein, mouse; D018398:Homeodomain Proteins; C546871:IBSP protein, human; C546872:Ibsp protein, mouse; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; D014157:Transcription Factors; D008156:Luciferases