Drosophila Dosage Compensation Loci Associate with a Boundary-Forming Insulator Complex

Abstract

Chromatin entry sites (CES) are 100- to 1,500-bp elements that recruit male-specific lethal (MSL) complexes to the X chromosome to upregulate expression of X-linked genes in male flies. CES contain one or more ∼20-bp GA-rich sequences called MSL recognition elements (MREs) that are critical for dosage compensation. Recent studies indicate that CES also correspond to boundaries of X-chromosomal topologically associated domains (TADs). Here, we show that an ∼1,000-kDa complex called the late boundary complex (LBC), which is required for the functioning of the Bithorax complex boundary , interacts specifically with a special class of CES that contain multiple MREs. Mutations in the MRE sequences of three of these CES that disrupt function abrogate interactions with the LBC. Moreover, reducing the levels of two LBC components compromises MSL recruitment. Finally, we show that several of the CES that are physically linked to each other are LBC interactors.

Complete reconstitution of bypass and blocking functions in a minimal artificialFab-7insulator fromDrosophila bithoraxcomplex

“…If so, inclusion of the Pita ×5 may facilitate LBC access. To test this possibility, we used chromatin immunoprecipitation (ChIP) data from embryos to examine the association of two proteins, CLAMP and GAF, which based on supershift and gel filtration experiments appear to be components of LBC (38,43,44). For CLAMP ChIP, we used antibodies directed against C-terminal sequences (Fig.…”

“…In the first approach, we generated mutations in dHS1 that compromise LBC binding. Previous studies on Fab-7 and on LBC recognition sequences elsewhere in the genome have implicated GAGAG motifs (and other GA-rich sequences) in LBC binding in vitro and in the functioning of these elements in vivo (38,43,44). The 240-bp dHS1 sequence contains four GAGAG motifs (designated 3, 4, 5, and 6 in SI Appendix, Fig.…”

“…Rather, the likely role of LBC is to provide a scaffold for the association of other, as-yet unknown factors. The reason for this is that LBC binds to sites elsewhere in the genome that have quite different functions (43,46). We would expect that the bypass activity of dHS1 might require not only an LBC association, but also other DNA-binding proteins.…”

“…Biochemical and genetic experiments have identified several factors that contribute to Fab-7 boundary function, including three multiprotein complexes-Elba, Insensitive, and LBCplus the architectural protein Pita. Elba and Insensitive bind to sites in HS*, HS1, and HS2 (38,(42)(43)(44), while Pita binds to two sites in HS2 (14,27,45). HS1 and HS3 contain recognition sequences for LBC (38,46).…”

“…However, optimal binding on EMSA is observed with larger DNAs, and the dHS1 sequences that can contribute to LBC binding (dHS1) span the distal half of this large hypersensitive site. Although the exact composition of LBC is unknown, antibody supershift experiments indicate that two DNA-binding proteins, GAF and CLAMP, are components of this complex (38,43).…”