SRP311796 PRJNA716544 GSE169410 The chromatin factor ROW cooperates with BEAF-32 in regulating long-range genes [MNase_seq_flyheads] Chromatin insulators are involved in transcription regulation and long-range chromatin contacts. In Drosophila the Boundary Element-Associated Factor Of 32kD (BEAF-32) binds to promoters of housekeeping genes and the boundaries of Topological Associated Domains (TAD) and is involved in the regulation of long-range targets by mechanisms that remain incompletely understood. Here we show that Relative-Of-WOC (ROW), which is part of the Heterochromatin Protein 1c (HP1c) complex, is essential for the long-range transcription regulation mediated by BEAF-32. We found that ROW physically interacts with the insulator complex proteins BEAF-32 and Chromator, as well as with HP1c, HP1b, and Without Children (WOC). Moreover, we found that ROW binds AT-rich sequences flanked by BEAF-32 motifs, which are located at promoters of housekeeping genes and in TAD boundaries. In agreement with these results, the genome distribution of ROW strongly correlated with BEAF-32 and WOC, but at a lower level with HP1c and HP1b. Knockdown of row resulted in downregulation of genes that are long-range targets of BEAF-32, are indirectly bound by BEAF-32 and ROW, and enriched with factors associated with RNA polymerase II pausing. These results suggest that ROW and BEAF-32 are involved in the recruitment of protein complexes required for transcription activation and long-range contacts between promoters of housekeeping and inducible genes. Overall design: Nucleosomes profile in Drosophila fly heads (triplicate). GSE169410 pubmed 36245419 parent_bioproject PRJNA716535