THE AT-RICH FLANKS OF THE OOCYTE-TYPE 5S RNA GENE OF XENOPUS-LAEVIS ACT AS A STRONG LOCAL SIGNAL FOR HISTONE H1-MEDIATED CHROMATIN REORGANIZATION IN-VITRO

In vivo, histone H1 plays an active role in establishing the transcrip tionally repressed chromatin state of the oocyte-type 5S RNA genes in the early stages of Xenopus development, By using fully defined in vit ro system of chromatin assembly on plasmids with cloned oocyte- or som atic-type 5S gene repeats we found that the oocyte repeat which compri ses a 120 bp oocyte-type 5S RNA gene placed within the few hundred bp long native AT-rich flanks, but not the somatic repeat (a similar 120 bp somatic-type 5S RNA gene placed within native CC-rich flanks) enabl es histone HI to realign the nucleosomal core particles densely packed on plasmid DNA, The realignment results in creation of the repeat uni t of similar to 240 bp and is achieved through complete removal of sev eral core histone complexes from plasmid template with the oocyte-type repeat. This effect of H1 is independent on the plasmid sequences and seems to be solely due to the presence in the oocyte-repeat of the AT -rich flanks, The effects of H1 are completely suppressed by distamyci n A, a drug that specifically recognizes and binds oligo(dA). oligo(dT ) runs in DNA, The binding of H1 results in increased protection of DN A sites within the AT-rich oocyte-type 5S repeat. In an in vitro trans cription assay performed with reconstituted chromatin templates contai ning plasmids with the oocyte- or somatic-type repeats only the transc ription of the oocyte-type 5S RNA gene was repressed in the presence o f physiological concentration of histone HI. These results support the view that the AT-rich flanks of the oocyte-type 5S RNA gene are invol ved in histone HI-mediated chromatin reorganization that results in th e transcriptional repression observed in vivo.