Identification of transcriptional targets of HOXA5.
The homeobox gene HOXA5 encodes a transcription factor that has been shown to play important roles in embryogenesis, hematopoiesis, and tumorigenesis. In order to decipher downstream signaling pathways of HOXA5, we utilized oligonucleotide microarray analysis to identify genes that are differentially expressed in HOXA5-induced cells compared with uninduced cells. Comparative analysis of gene expression changes after 9 h of HOXA5 induction in Hs578T breast cancer cells identified 306 genes whose expression was modulated at least 2-fold. Ten of these 306 genes were also up-regulated by at least 2-fold at 6 h post-induction. The expression of all of these 10 genes was confirmed by semiquantitative reverse transcription-PCR. Among these 10 genes, which are most likely to be direct targets of HOXA5, we initiated an investigation into the pleiotrophin gene by first cloning its promoter. Transient transfection assays indicated that HOXA5 can specifically activate the pleiotrophin promoter. Promoter deletion, chromatin immunoprecipitation assay, and gel-shift assays were performed to show that HOXA5 can directly bind to one binding site on the pleiotrophin promoter. These data strongly suggest that microarray analysis can successfully identify many potential direct downstream genes of HOXA5. Further functional analysis of these targets will allow us to better understand the diverse functions of HOXA5 in embryonic development and tumorigenesis.
Chen
Hexin
H
Breast Cancer Program, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Hospital, Baltimore, Maryland 21231-1000, USA.
Rubin
Ethel
E
Zhang
Huiping
H
Chung
Seung
S
Jie
Charles C
CC
Garrett
Elizabeth
E
Biswal
Shyam
S
Sukumar
Saraswati
S
D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D016207:Cytokines / Q000378:metabolism; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D010750:Phosphoproteins / Q000378:metabolism* / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
D002352:Carrier Proteins; D016207:Cytokines; C489213:HOXA5 protein, human; D018398:Homeodomain Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; C066845:pleiotrophin; D001616:beta-Galactosidase