2 DISTINCT TYPES OF REPRESSION DOMAIN IN ENGRAILED - ONE INTERACTS WITH THE GROUCHO COREPRESSOR AND IS PREFERENTIALLY ACTIVE ON INTEGRATED TARGET GENES

Active transcriptional repression has been characterized as a function of many regulatory factors. It facilitates combinatorial regulation o f gene expression by allowing repressors to be dominant over activator s under certain conditions. Here, we show that the Engrailed protein u ses two distinct mechanisms to repress transcription, One activity is predominant under normal transient transfection assay conditions in cu ltured cells. A second activity is predominant in an in vivo active re pression assay. The domain mediating the in vivo activity (eh1) is hig hly conserved throughout several classes of homeoproteins and interact s specifically with the Groucho corepressor, While eh1 shows only weak activity in transient transfections, much stronger activity is seen i n culture when an integrated target gene is used. In this assay, the r elative activities of different repression domains closely parallel th ose seen in vivo, with eh1 showing the predominant activity. Reducing the amounts of repressor and target gene in a transient transfection a ssay also increases the sensitivity of the assay to the Groucho intera ction domain, albeit to a lesser extent. This suggests that it utilize s rate-limiting components that are relatively low in abundance. Since Groucho itself is abundant in these cells, the results suggest that a limiting component is recruited effectively by the repressor-corepres sor complex only on integrated target genes.