An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene.
Tissue and cell-type specific expression of the rat osteocalcin (rOC) gene involves the interplay of multiple transcriptional regulatory factors. In this report we demonstrate that AML-1 (acute myeloid leukemia-1), a DNA-binding protein whose genes are disrupted by chromosomal translocations in several human leukemias, interacts with a sequence essential for enhancing tissue-restricted expression of the rOC gene. Deletion analysis of rOC promoter-chloramphenicol acetyltransferase constructs demonstrates that an AML-1-binding sequence within the proximal promoter (-138 to -130 nt) contributes to 75% of the level of osteocalcin gene expression. The activation potential of the AML-1-binding sequence has been established by overexpressing AML-1 in osteoblastic as well as in nonosseous cell lines. Overexpression not only enhances rOC promoter activity in osteoblasts but also mediates OC promoter activity in a nonosseous human fibroblastic cell line. A probe containing this site forms a sequence specific protein-DNA complex with nuclear extracts from osteoblastic cells but not from nonosseous cells. Antisera supershift experiments indicate the presence of AML-1 and its partner protein core-binding factor beta in this osteoblast-restricted complex. Mutations of the critical AML-1-binding nucleotides abrogate formation of the complex and strongly diminish promoter activity. These results indicate that an AML-1 related protein is functional in cells of the osteoblastic lineage and that the AML-1-binding site is a regulatory element important for osteoblast-specific transcriptional activation of the rOC gene.
Banerjee
C
C
Department of Cell Biology and Cancer Center, University of Massachusetts Medical Center, Worcester, 01655, USA.
Hiebert
S W
SW
Stein
J L
JL
Lian
J B
JB
Stein
G S
GS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D016384:Consensus Sequence; D050676:Core Binding Factor Alpha 2 Subunit; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation
D050676:Core Binding Factor Alpha 2 Subunit; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; C493728:RUNX1 protein, human; D011994:Recombinant Proteins; C493727:Runx1 protein, rat; D014157:Transcription Factors; D015675:Osteocalcin; D004247:DNA