Packaging of DNA into nucleosomes and the formation of higher-order chromatin structures determine DNA accessibility and activity of genome domains. We identified an RNA-dependent mechanism maintaining the open chromatin structure within euchromatic regions in Drosophila cells. The mechanism of reversible chromatin opening, reconstituted in vitro, depends on the Drosophila decondensation factor 31 (Df31) that specifically binds to RNA and localizes to euchromatic regions. Df31 is capable to tether a heterogeneous pool of short, single-stranded RNAs to chromatin. This class of chromatin-associated RNA (caRNA) is stably linked to chromatin and is largely composed of snoRNAs, which are preferentially bound by Df31. We suggest that the Df31-mediated linkage of snoRNAs and chromatin, forms a RNA-chromatin network resulting in the establishment of open chromatin domains. Analysis of caRNAs in human cells also reveals a strong enrichment of snoRNAs, implying a conserved role for these molecules in higher-order structures of chromatin.