A NUCLEOSOME POSITIONED IN THE DISTAL PROMOTER REGION ACTIVATES TRANSCRIPTION OF THE HUMAN U6 GENE

To investigate the consequences of chromatin reconstitution for transc ription of the human U6 gene, we assembled nucleosomes on both plasmid s and linear DNA fragments containing the U6 gene, Initial experiments with DNA fragments revealed that U6 sequences located between the dis tal sequence element (DSE) and the proximal sequence element (PSE) lea d to the positioning of a nucleosome partially encompassing these prom oter elements, Furthermore, indirect end-labelling analyses of the rec onstituted U6 wild-type plasmids showed strong micrococcal nuclease cu ts near the DSE and PSE, indicating that a nucleosome is located betwe en these elements, To investigate the influence that nucleosomes exert on U6 transcription, we used two different experimental approaches fo r chromatin reconstitution, both of which resulted in the observation that transcription of the U6 wild-type gene was enhanced after chromat in assembly, To ensure that the facilitated transcription of the nucle osomal templates is in fact due to a positioned nucleosome, we constru cted mutants of the U6 gene in which the sequences between the DSE and PSE were progressively deleted, In contrast to what was observed with the wild-type genes, transcription of these deletion mutants was sign ificantly inhibited when they were packaged into nucleosomes.