INHIBITION OF RETINOIC ACID-INDUCED ACTIVATION OF 3' HUMAN HOXB GENESBY ANTISENSE OLIGONUCLEOTIDES AFFECTS SEQUENTIAL ACTIVATION OF GENES LOCATED UPSTREAM IN THE 4 HOX CLUSTERS

Most homeobox genes belonging to the Hox family are sequentially activ ated in embryonal carcinoma cells upon treatment with retinoic acid. G enes located at the 3' end of each one of the four Hox clusters are ac tivated first, whereas upstream Hox genes are activated progressively later. This activation has been extensively studied for human HOX gene s in the NT2/D1 cell line and shown to take place at the transcription al level. To understand the molecular mechanisms of sequential HOX gen e activation in these cells, we tried to modulate the expression of 3' HOX genes through the use of antisense oligonucleotides added to the culture medium. We chose the HOXB locus. A 5- to 15-fold reduction of the expression of HOXB1 and HOXB3 was sufficient to produce a signific ant inhibition of the activation of the upstream HOXB genes, as well a s of their paralogs in the HOXA, HOXC, and HOXD clusters. Conversely, no effect was detectable on downstream HOX genes. The extent of this i nhibition increased for progressively more-5' genes. The stability of the corresponding mRNAs appeared to be unaffected, supporting the idea that the observed effect might be mediated at the transcriptional lev el. These data suggest a cascade model of progressive activation of Ho x genes, with a 3'-to-5' polarity.